Hla-a1,-a2,-a3,-a24,-b7, and-b44 tumor associated antigen peptides and compositions

ABSTRACT

A peptide or composition comprising at least one epitope or analog from CEA, HER2/neu, MAGE2, MAGE3, or p53.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to the field of biology. In a particular embodiment, it relates to peptides, polynucleotides, and compositions useful to monitor or elicit an immune response to selected tumor-associated antigens.

2. Related Art

The field of immunotherapy is yielding new approaches for the treatment of cancer, including the development of improved cancer vaccines (Krul, K. G., Decision Resources, 10.1-10.25 (1998)). While vaccines provide a mechanism of directing immune responses towards the tumor cells, there are a number of mechanisms by which tumor cells circumvent immunological processes (Pardoll, D. M., Nature Medicine (Vaccine Supplement), 4:525-531 (1998)). Recent advances indicate that the efficacy of peptide vaccines may be increased when combined with approaches which enhance the stimulation of immune responses, such as the use of Interleukin-2 or autologous dendritic cells (DC) (Abbas et al., eds., Cellular and Molecular Immunology, 3^(rd) Edition, W.B. Saunders Company, pub. (1997)).

In a Phase I study, Murphy, et al., demonstrated that Human Leukocyte Antigen (HLA)-A2-binding peptides corresponding to sequences present in prostate specific antigen (PSA) stimulated specific cytotoxic T-cell lymphocyte (CTL) responses in patients with prostate cancer (Murphy et al., The Prostate 29:371-380 (1996)). Recently, Rosenberg, et al., evaluated the safety and mechanism of action of a synthetic HLA-A2 binding peptide derived from the melanoma associated antigen, gp100, as a cancer vaccine to treat patients with metastatic melanoma Rosenberg et al., Nature Med., 4:321-327 (1998)). Based on immunological assays, 91% of patients were successfully immunized with the synthetic peptide. In addition, 42% (13/31) of patients who received the peptide vaccine in combination with IL-2 treatment, demonstrated objective cancer responses. Finally, Nestle, et al., reported the vaccination of 16 melanoma patients with peptide- or tumor lysate-pulsed DC (Nestle et al., Nature Med 4:328-332 (1998)). Peptide-pulsed DC induced immune responses in (11/12) patients immunized with a vaccine comprised of 1-2 peptides. Objective responses were evident in 5/16 (3 peptide-pulsed, 2 tumor-lysate pulsed) evaluated patients in this study. These Phase I safety studies provided evidence that HLA-A2 binding peptides of known tumor-associated antigens demonstrate the expected mechanism of action. These vaccines were generally safe and well tolerated. Vaccine molecules related to four cancer antigens, CEA, HER2/neu, MAGE2, and, MAGE3 have been disclosed. (Kawashima et al., Human Immunology, 59:1-14 (1998))

Preclinical studies have shown that vaccine-pulsed DC mediate anti-tumor effects through the stimulation of antigen-specific CTL (Mandelboim et al, Nature Med., 1: 1179-1183 (1995); Celluzzi et al, J Exp Med 183:283-287 (1996); Zitvogel et al., J Exp Med 183:87-97 (1996); Mayordomo et al, Nature Med 1:1297-1302 (1995)). CTL directly lyse tumor cells and also secrete an array of cytokines such as interferon gamma (IFNγ), tumor necrosis factor (TNF) and granulocyte-macrophage colony stimulating factor (GM-CSF), that further amplify the immune reactivity against the tumor Tells. CTL recognize tumor associated antigens (TAA) in the form of a complex composed of 8-11 amino acid residue peptide epitopes, bound to Major Histocompatibility Complex (MHC) molecules (Schwartz, B. D., The human major histocompatibility complex HL in basic & clinical immunology Stites et al., eds., Lange Medical Publication: Los Altos, pp. 52-64, 4^(th) ed.). Peptide epitopes are generated through intracellular processing of proteins. The processed peptides bind to newly synthesized MHC molecules and the epitope-MHC complexes are expressed on the cell surface. These epitope-MHC complexes are recognized by the T cell receptor of the CTL. This recognition event is required for the activation of CmL as well as induction of the effector functions such as lysis of the target tumor cell.

MHC molecules are highly polymorphic proteins that regulate T cell responses (Schwartz, B. D., The human major histocomnpatibility complex SLA in basic & clinical immunology Stites et al., eds., Lange Medical Publication: Los Altos, pp. 52-64, 4^(th) ed.). The species-specific MHC homologues that display CTL epitopes in humans are termed HLA. HLA class I molecules can be divided into several families or “supertypes” based upon their ability to bind similar repertoires of peptides. Vaccines which bind to HLA supertypes such as A2, A3, and B7, will afford broad, non-ethnically biased population coverage. As seen in Table 11, population coverage is 84-90% for varous ethnicities, with an average coverage of the sample ethnicities at 87%.

One of the main factors contributing to the dynamic interplay between host and disease is the immune response mounted against the pathogen, infected cell, or malignant cell. In many conditions such immune responses control the disease. Several animal model systems and prospective studies of natural infection in humans suggest that immune responses against a pathogen can control the pathogen, prevent progression to severe disease and/or eliminate the pathogen. A common theme is the requirement for a multispecific T cell response, and that narrowly focused responses appear to be less effective.

In the cancer setting there are several findings that indicate that immune responses can impact neoplastic growth:

First, the demonstration in many different animal models, that anti-tumor T cells, restricted by MHC class I, can prevent or treat tumors.

Second, encouraging results have come from immunotherapy trials.

Third, observations made in the course of natural disease correlated the type and composition of T cell infiltrate within tumors with positive clinical outcomes (Coulie P G, et al. Antitumor immunity at work in a melanoma patient In Advances in Cancer Research, 213-242, 1999).

Finally, tumors commonly have the ability to mutate, thereby changing their immunological recognition. For example, the presence of monospecific CTL was also correlated with control of tumor growth, until antigen loss emerged (Riker A, et al., Immune selection after antigen-specific immunotherapy of melanoma Surgery, Aug: 126(2):112-20, 1999; Marchand M, et al., Tumor regressions observed in patients with metastatic melanoma treated with an antigenic peptide encoded by gene MAGE-3 and presented by HLA-A1 Int. J. Cancer 80(2):219-30, Jan. 18, 1999). Similarly, loss of beta 2 microglobulin was detected in 5/13 lines established from melanoma patients after receiving immunotherapy at the NCI (Restifo N P, et al., Loss of functional Beta2—microglobulin in metastatic melanomas from five patients receiving immunotherapy Journal of the National Cancer Institute, Vol. 88 (2), 100-108, January 1996). It has long beeni recognized that HLA class I is frequently altered in various tumor types. This has led to a hypothesis that this phenomenon might reflect immune pressure exerted on the tumor by means of class I restricted CTL. The extent and degree of alteration in HLA class I expression appears to be reflective of past immune pressures, and may also have prognostic value (van Duinen SG, et al., Level of HLA antigens in locoregional metastases and clinical course of the disease in patients with melanoma Cancer Research 48, 1019-1025, February 1988; Möller P, et al., Influence of major histocompatibility complex class I and II antigens on survival in colorectal carcinoma Cancer Research 51, 729-736, January 1991). Taken together, these observations provide a rationale for immunotherapy of cancer and infectious disease, and suggest that effective strategies need to account for the complex series of pathological changes associated with disease.

The frequency of alterations in class I expression is the subject of numerous studies (Algarra I, et al., The HLA crossroad in tumor immunology Human Immunology 61, 65-73, 2000). Rees and Mian estimate allelic loss to occur overall in 3-20% of tumors, and allelic deletion to occur in 15-50% of tumors. It should be noted that each cell carries two separate sets of class I genes, each gene carrying one HLA-A and one HLA-B locus. Thus, fully heterozygous individuals carry two different HLA-A molecules and two different HLA-B molecules. Accordingly, the actual frequency of losses for any specific allele could be as little as one quarter of the overall frequency. They also note that, in general, a gradient of expression exists between normal cells, primary tumors and tumor metastasis. In a study from Natali and coworkers (Natali P G, et al., Selective changes in expression of HLA class I polymorphic determinants in human solid tumors PNAS USA 86:6719-6723, September 1989), solid tumors were investigated for total HLA expression, using W6/32 antibody, and for allele-specific expression of the A2 antigen, as evaluated by use of the BB7.2 antibody. Tumor samples were derived from primary cancers or metastasis, for 13 different tumor types, and scored as negative if less than 20%, reduced if in the 30-80% range, and normal above 80%. All tumors, both primary and metastatic, were HLA positive with W6/32. In terms of A2 expression, a reduction was noted in 16.1% of the cases, and A2 was scored as undetectable in 39.4% of the cases. Garrido and coworkers (Garrido F, et al., Natural history of HLA expression during tumour development Immunol Today 14(10):491-99, 1993) emphasize that HLA changes appear to occur at a particular step in the progression from benign to most aggressive. Jiminez et al (Jiminez P, et al., Microsatellite instability analysis in tumors with different mechanisms for total loss of HLA expression. Cancer Immunol Immunother 48:684-90, 2000) have analyzed 118 different tumors (68 colorectal, 34 laryngeal and 16 melanomas). The frequencies reported for total loss of HLA expression were 11% for colon, 18% for melanoma and 13% for larynx. Thus, HLA class I expression is altered in a significant fraction of the tumor types, possibly as a reflection of immune pressure, or simply a reflection of the accumulation of pathological changes and alterations in diseased cells.

A majority of the tumors express HLA class I, with a general tendency for the more severe alterations to be found in later stage and less differentiated tumors. This pattern is encouraging in the context of immunotherapy, especially considering that: 1) the relatively low sensitivity of immunohistochemical techniques might underestimate HLA expression in tumors; 2) class I expression can be induced in tumor cells as a result of local inflammation and lymphokine release; and, 3) class I negative cells are sensitive to lysis by NK cells.

Recent evidence has shown that certain patients infected with a pathogen, whom are initially treated with a therapeutic regimen to reduce pathogen load, have been able to maintain decreased pathogen load when removed from the therapeutic regimen, i.e., during a “drug holiday” (Rosenberg, B., et al., Immune control of HIV-1 after early treatment of acute infection Nature 407:523-26, Sep. 28, 2000) As appreciated by those skilled in the art, many therapeutic regimens for both pathogens and cancer have numerous, often severe, side effects. During the drug holiday, the patient's immune system is keeping the disease in check.

Various approaches have, or are, being employed as cancer vaccines. Table 1 overviews the major cancer vaccine approaches and the various advantages and disadvantages of each.

Currently there are a number of unmet needs in the area of cancer treatment. This is evidenced by the side effects associated with existing therapies employed for cancer treatment and the fact that less than 50% of patients are cured by current therapies. Therefore, an opportunity exists for a product with the ability to either increase response rates, duration of response, overall survival, disease free survival or quality of life.

SUMMARY OF THE INVENTION

In some embodiments, the invention is directed to an isolated peptide comprising or consisting of one or more HLA-A1, -A3, -A24, -B7, and/or B44 epitopes and/or HLA-A1, -A2, -A3, -A24, -B7, and/or B44 analogs. The peptide may comprise mutiple epitopes and/or analogs, and may comprise additional amino acids, including other CTL epitopes, HTL epitopes, linkers, spacers, carriers, etc.

In further embodiments, the invention is directed to polynucleotides encoding such peptides.

In further embodiments, the invention is directed to a composition comprising one or more of the above peptides and/or polynucleotides and one or more additional components. Additional components include diluents, excipients, CTL epitopes, HTL epitopes, carriers, liposomes, HLA heavy chains, β2-microglobulin, strepavidin, antigen-presenting cells, adjuvants, etc.

In further embodiments, the invention is directed to prophylactic, therapeutic, diagnostic, and prognostic methods using the peptides, polynucleotides, and compositions of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES

FIG. 1 depicts that PADRE promotes antigen specific T cell responses from human PBMC. In FIG. 1, PBMC from three healthy donors (donors 431, 397, and 344) were stimulated in vitro. In brief, Ficoll-Paque (Pharmacia LKB) purified PBMC were plated at 4×10⁶ cells/well in a 24-well tissue culture plate (Costar). The peptides were added at a final concentration of 10 μg/ml and incubated at 37° C. for 4 days. Recombinant interleukin-2 was added at a final concentration of 10 ng/ml and the cultures were fed every three days with fresh media and cytokine. Two additional stimulations of the T cells with antigen were performed on approximately days 14 and 28. The T cells (3×10⁵ cells/well) were restimulated with 10 μg/ml peptide using irradiated (7500 rads) autologous PBMC cells. T cell proliferative responses were determined using a ³H-thymidine incorporation assay.

FIG. 2 depicts that PADRE-specific proliferative responses are induced via peptide vaccination. In FIG. 2, two weeks after vaccination, PBMC of 4 out of 12 cervical cancer patients (002, 005, 008, and 014) displayed proliferation when stimulated in vitro with 5 μg/ml PADRE peptide (4/12=33% responding patients, 95% interval 10-65%) (Tx=treatment). The proliferation index of multiple wells was calculated as the mean cpm from experimental wells divided by the mean cpm from control wells. PADRE-specific responses were considered positive when the proliferation index exceeded 5. The variation between replicates was always less than 25% (Messing et al., “Detection of T helper responses, but not of human papillomavirus-specific cytotoxic T lymphicyte responses, after peptide vaccination of captients with cervical carcinoma,” J Immuother 23(2):255-66 (March-April 2000)).

FIG. 3 depicts that splenic DC from ProGP-treated mice present HBV-derived CTL epitopes to a CTL line. In FIG. 3, Splenic DC from ProGP-treated HLA-A2.1/K^(b)-H-2^(bxs) transgenic mice (33 μg/animal, QD, SC for 7 days) were enriched using an anti-CD11c antibody (Miltenyi Biotec). B cells were isolated from normal spleen by magnetic separation after treating cells with biotinylated anti-CD19 antibody and Strepavidin-coupled beads (Miltenyi Biotec). DC were also generated from bone marrow cells by culture with GM-CSF/IL-4. DC or B cells, (1×10⁵ cells) were incubated with 1×10⁴ CTL line 1168 and varying concentrations of the HBV Pol 455 peptide in Opti-MEM I medium containing 3 μg/ml (32-microglobulin (Scripps Laboratories). Cells were added to 96-flat bottom well ELISA plates that were pre-coated with an anti-IFNγ capture antibody. After incubation for 18-20 hr at 37° C., in situ production of IFNγ by stimulated line 1168 was measured using a sandwich ELISA. Data shown is from one experiment. Similar results have been obtained in additional experiments. Studies were performed at Epimmune Inc., San Diego, Calif.

FIG. 4 depicts that splenic DC from ProGP-treated mice induce CTL responses in vivo. In FIG. 4, Splenic DC from ProGP treated HLA-A2.1 transgenic mice (33 μg/mouse, QD, SC for 7 days) were pulsed in vitro with HBV Pol 455 peptide (10⁶ cell per ml peptide at 10 μg/ml) in Opti-MEM I medium (Gibco Life Sciences) containing 3 μg/ml β2-microglobulin (Scripps Laboratories). After peptide pulsing for 3 hr at room temperature, DC were washed twice and 10⁶ cells were injected IV into groups of three transgenic mice. Epitope-pulsed GM-CSF/IL-4 expanded DC and “mock-pulsed” ProGP derived DC were also tested for comparison. Seven days after receiving the primary immunization with DC, animals were boosted with the same DC populations. At fourteen days after the primary immunization, spleen cells from immunized animals were restimulated twice in vitro in the presence of the Pol 455 peptide. CTL activity following restimulations was measured using a standard ⁵¹Cr release assay in which the lysis of ⁵¹Cr-labeled HLA-A2.1-transfected Jurkat target cells was measured in the presence (circle symbols) or absence of peptide (square symbols). The data points shown in Panels A-C represent a composite of lytic activity from a triplicate set of cultures. Panel A, splenic DC from ProGP (SD-9427) treated animals pulsed with the HBV Pol 455 peptide. Panel B, GM-CSF/IL-4 expanded DC pulsed with HBV Pol 455 peptide. Panel C, mock-pulsed DC from ProGP treated animals. Studies were performed at Epimmune Inc., San Diego, Calif.

FIG. 5 presents a schematic of a dendritic cell pulsing and testing procedure.

FIG. 6 shows that CEA.241K10-specific CTLs recognize analog and wildtype peptide-pulsed targets. Individual cultures were tested against EHM without peptide (open bar), EHM pulsed with CEA.241K10 (hatched bar) and with EHM pulsed with CEA.241 (solid bar). A positive response was 50 pg/well above background and twice background. Well number 48 is negative and is included only for comparison.

FIG. 7 shows that p53.172B5K10-specific CTLs recognize analog and wildtype peptide-pulsed targets and transfected tumor target cells. Individual cultures were tested against EHM without peptide (open bar), EHM pulsed with p53.172B5K10 (hatched bar), and EHM pulsed with p53.172 (solid bar), SW403 (A3+/p53−, dotted bar) or SW403 transfected with p53 (A3+/p53+, crosshatched bar). A positive response was defined as one in which the specific lysis (sample—background) was 10% or higher. Well number 1 is negative and is included only for comparison.

DETAILED DESCRIPTION OF THE INVENTION

This invention provides peptides that can be used to monitor an immune response to a tumor associated antigen or to create a cancer vaccine that stimulates the cellular arm of the immune system, especially when one or more peptides are combined. In particular embodiments, compositions mediate immune responses against tumors in individuals who bear at least one allele of HLA-A1, HLA-A1 supertype, and/or HLA-A2, HLA-A2 supertype, and/or HLA-A3, HLA-A3 supertype, and/or HLA-A24, HLA-A24 supertype, and/or, -B7, -B7 supertype, and/or -B44, -B44 supertype (see Table 5 for a listing of the members of these and other supertypes and types); such compositions will generally be referred to as A1, A2, A3, A24, B7, or B44, compositions (or combinations thereof).

An A2, A3, B7, A24, A1, and/or B44 composition may, for example, act as a vaccine to stimulate the immune system to recognize and kill tumor cells, leading to increased quality of life, and/or disease-free or overall survival rates for patients treated for cancer. In a preferred embodiment, a composition of the invention such as a vaccine will be administered to HLA-A2 or HLA-A2 supertype, HLA-A3 or HLA-A3 supertype, -B7 or -B7 supertype, B-44 or -B44 supertype, -A24 or -A1 positive individuals who have a cancer that expresses at least one of the TAAs from which the epitopes or analogs were selected (e.g., CEA, p53, HER2/neu, MAGE2/3), examples of such cancers being breast, colon, lung, and gastric cancers and for MAGE 2/3, some melanomas. Alternative embodiments of a vaccine are directed at patients who bear additional HLA alleles, or who do not bear an A2, A3, B7, A24, B44, and/or A1 allele at all. Thereby, an A2, A3, B7, A24, B44, and/or A1 vaccine improves the standard of care for patients being treated for breast, colon, lung, or gastric cancers, or melanoma.

The peptides and corresponding nucleic acids and compositions of the present invention are useful for stimulating an immune response to TAAs by stimulating the production of CTL and optionally HTL responses, e.g. therapeutic prophylaxis, and are also useful for monitoring an immune response, e.g., diagnosis and prognosis. The peptides, which contain A2, A3, B7, A24, A1 and/or B44 epitopes derived directly or indirectly (i.e. by analoging) from native TAA protein amino acid sequences, are able to bind to HLA molecules and stimulate an immune response to TAAs. The complete sequence of the TAAs proteins to be analyzed can be obtained from GenBank. See Table 25.

The epitopes of the invention have been identified in a number of ways, as will be discussed below. Also discussed in greater detail is that analogs have been derived in which the binding activity for HLA molecules was modulated by modifying specific amino acid residues to create analogs which exhibit altered (e.g., improved) immunogenicity. Further, the present invention provides peptides, polynucleotides, and compositions that are capable of interacting with HLA molecules encoded by various genetic alleles to provide broader population coverage than prior compositions, for prophylaxis, therapy, diagnosis, prognosis, etc.

Definitions

The invention can be better understood with reference to the following definitions:

Throughout this disclosure, “binding data” results are often expressed in terms of “IC₅₀'s.” IC₅₀ is the concentration of peptide in a binding assay at which 50% inhibition of binding of a reference peptide is observed. Given the conditions in which the assays are run (i.e., limiting HLA proteins and labeled peptide concentrations), these values approximate K_(D) values. Assays for determining binding are described in detail, e.g., in PCT publications WO 94/20127 and WO 94/03205, and other publications such Sidney et al., Current Protocols in Immunology 18.3.1 (1998); Sidney, et al., J. Immunol. 154:247 (1995); and Sette, et al., Mol. Immunol. 31:813 (1994). It should be noted that IC₅₀ values can change, often dramatically, if the assay conditions are varied, and depending on the particular reagents used (e.g., HLA preparation, etc.). For example, excessive concentrations of HLA molecules will increase the apparent measured IC₅₀ of a given ligand.

Alternatively, binding is expressed relative to a reference peptide. Although as a particular assay becomes more, or less, sensitive, the IC₅₀'s of the peptides tested may change somewhat, the binding relative to the reference peptide will not significantly change. For example, in an assay run under conditions such that the IC₅₀ of the reference peptide increases 10-fold, the IC₅₀ values of the test peptides will also shift approximately 10-fold. Therefore, to avoid ambiguities, the assessment of whether a peptide is a good (i.e. high), intermediate, weak, or negative binder is generally based on its IC₅₀, relative to the IC₅₀ of a standard peptide. The Tables included in this application present binding data in a preferred biologically relevant form of IC₅₀ nM.

Binding may also be determined using other assay systems including those using: live cells (e.g., Ceppellini et al., Nature 339:392 (1989); Christnick et al., Nature 352:67 (1991); Busch et al., Int. Immunol. 2:443 (1990); Hill et al., J. Immunol. 147:189 (1991); del Guercio et al., J. Immunol. 154:685 (1995)), cell free systems using detergent lysates (e.g., Cerundolo et al., J. Immunol. 21:2069 (1991)), immobilized purified MHC (e.g. Hill et al., J. Immunol. 152, 2890 (1994); Marshall et al., J. Immunol. 152:4946 (1994)), ELISA systems (e.g., Reay et al., EMBO J. 11:2829 (1992)), surface plasmon resonance (e.g., Khilko et al., J. Biol. Chem. 268:15425 (1993)); high flux soluble phase assays (Hammer et al., J. Exp. Med. 180:2353 (1994)), and measurement of class I MHC stabilization or assembly (e.g., Ljunggren et al., Nature 346:476 (1990); Schumacher et al., Cell 62:563 (1990); Townsend et al., Cell 62:285 (1990); Parker et al., J. Immunol. 149:1896 (1992)).

As used herein, “high affinity” with respect to HLA class I molecules is defined as binding with an IC₅₀ or K_(D) value, of 50 nM or less, “intermediate affinity” is binding with an IC₅₀ or K_(D) value of between 50 and about 500 nM, weak affinity is binding with an IC₅₀ or K_(D) value of between about 500 and about 5000 nM. “High affinity” with repect to binding to HLA class II molecules is defined as binding with an IC₅₀ or K_(D) value of 100 nM or less; “intermediate affinity” is binding with an IC₅₀ or K_(D) value of between about 100 and about 1000 nM.

A “computer” or “computer system” generally includes: a processor and related computer programs; at least one information storage/retrieval apparatus such as a hard drive, a disk drive or a tape drive; at least one input apparatus such as a keyboard, a mouse, a touch screen, or a microphone; and display structure, such as a screen or a printer. Additionally, the computer may include a communication channel in communication with a network. Such a computer may include more or less than what is listed above.

“Cross-reactive binding” indicates that a peptide is bound by more than one HLA molecule; a synonym is degenerate binding.

A “cryptic epitope” elicits a response by immunization with an isolated peptide, but the response is not cross-reactive in vitro when intact whole protein, which comprises the epitope, is used as an antigen.

The term “derived” when used to discuss an epitope is a synonym for “prepared.” A derived epitope can be isolated from a natural source, or it can be synthesized in accordance with standard protocols in the art. Synthetic epitopes can comprise artificial amino acids “amino acid mimetics,” such as D isomers of natural occurring L amino acids or non-natural amino acids such as cyclohexylalanine. A derived/prepared epitope can be an analog of a native epitope.

A “diluent” includes sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred diluent for pharmaceutical compositions. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as diluents, particularly for injectable solutions.

A “dominant epitope” is an epitope that induces an immune response upon immunization with a whole native antigen (see, e.g., Sercarz, et al., Annu. Rev. Immunol. 11:729-766, 1993). Such a response is cross-reactive in vitro with an isolated peptide epitope.

An “epitope” is the collective features of a molecule, such as primary, secondary and tertiary peptide structure, and charge, that together form a site recognized by an immunoglobulin, T cell receptor or HLA molecule. Alternatively, an epitope can be defined as a set of amino acid residues which is involved in recognition by a particular immunoglobulin, or in the context of T cells, those residues necessary for recognition by T cell receptor proteins and/or Major Histocompatibility Complex (MHC) receptors. Epitopes are present in nature, and can be isolated, purified or otherwise prepared/derived by humans. For example, epitopes can be prepared by isolation from a natural source, or they can be synthesized in accordance with standard protocols in the art. Synthetic epitopes can comprise artificial amino acids, “amino acid milmetics,” such as D isomers of naturally-occurring L amino acids or non-naturally-occuring amino acids such as cyclohexylalanine. Throughout this disclosure, epitopes may be referred to in some cases as peptides. The epitopes and analogs of the invention are set forth in Tables 16A-23 and B44 Table.

It is to be appreciated that proteins or peptides that comprise an epitope or an analog of the invention as well as additional amino acid(s) are still within the bounds of the invention. In certain embodiments, the peptide comprises a fragment of an antigen. A “fragment of an antigen” or “antigenic fragment” or simply “fragment” is a portion of an antigen which has 100% identity with a wild type antigen or naturally-ocurring variant thereof. The fragment may or may not comprise an epitope of the invention. The fragment may be less than or equal to 600 amino acids, less than or equal to 500 amino acids, less than or equal to 400 amino acids, less than or equal to 250 amino acids, less than or equal to 100 amino acids, less than or equal to 85 amino acids, less than or equal to 75 amino acids, less than or equal to 65 amino acids, or less than or equal to 50 amino acids in length. In certain embodiments, a fragment is e.g., less than 101 or less than 51 amino acids in length, in any increment down to 5 amino acids in length. For example, the fragment may be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 amino acids in length.

In certain embodiments, there is a limitation on the length of a peptide of the invention. The embodiment that is length-limited occurs when the protein/peptide comprising an epitope of the invention comprises a region (i.e., a contiguous series of amino acids) having 100% identity with a native sequence. In order to avoid the definition of epitope from reading, e.g., on whole natural molecules, there is a limitation on the length of any region that has 100% identity with a native peptide sequence. Thus, for a peptide comprising an epitope of the invention and a region with 100% identity with a native peptide sequence, the region with 100% identity to a native sequence generally has a length of: less than or equal to 600 amino acids, often less than or equal to 500 amino acids, often less than or equal to 400 amino acids, often less than or equal to 250 amino acids, often less than or equal to 100 amino acids, often less than or equal to 85 amino acids, often less than or equal to 75 amino acids, often less than or equal to 65 amino acids, and often less than or equal to 50 amino acids. In certain embodiments, an “epitope” of the invention is comprised by a peptide having a region with less than 51 amino acids that has 100% identity to a native peptide sequence, in any increment down to 5 amino acids.

Accordingly, peptide or protein sequences longer than 600 amino acids are within the scope of the invention, so long as they do not comprise any contiguous sequence of more than 600 amino acids that have 100% identity with a native peptide sequence. For any peptide that has five contiguous residues or less that correspond to a native sequence, there is no limitation on the maximal length of that peptide in order to fall within the scope of the invention. It is presently preferred that a peptide of the invention (e.g. a peptide comprising an epitope of the invention) be less than 600 residues long in any increment down to eight amino acid residues.

“Human Leukocyte Antigen” or “HLA” is a human class I or class II Major Histocompatibility Complex (MHC) protein (see, e.g., Stites, et al., IMMUNOLOGY, 8^(TH) ED., Lange Publishing, Los Altos, Calif. (1994).

An “HLA supertype or HLA family”, as used herein, describes sets of HLA molecules grouped on the basis of shared peptide-binding specificities. HLA class I molecules that share somewhat similar binding affinity for peptides bearing certain amino acid motifs are grouped into such HLA supertypes. The terms HLA superfamily, HLA supertype family, HLA family, and HLA xx-like molecules (where “xx” denotes a particular HLA type), are synonyms. See Tables 14-23 plus B44 Table.

As used herein, “high affinity” with respect to HLA class I molecules is defined as binding with an IC₅₀, or K_(D) value, of 50 nM or less; “intermediate affinity” is binding with an IC₅₀ or K_(D) value of between about 50 and about 500 nM; “weak affinity” is binding with an IC₅₀ or K_(D) value between about 500 and about 5000 nM. “High affinity” with respect to binding to HLA class II molecules is defined as binding with an IC₅₀ or K_(D) value of 100 nM or less; “intermediate, affinity” is binding with an IC₅₀ or K_(D), value of between about 100 and about 1000 nM. See “binding data.”

An “IC₅₀” is the concentration of peptide in a binding assay at which 50% inhibition of binding of a reference peptide is observed. Given the conditions in which the assays are run (i.e., limiting HLA proteins and labeled peptide concentrations), these values approximate K_(D) values. See “binding data.”

The terms “identical” or percent “identity,” in the context of two or more peptide sequences or antigen fragments, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues that are the same, when compared and aligned for maximum correspondence over a comparison window, as measured using a sequence comparison algorithm or by manual alignment and visual inspection.

An “immunogenic” peptide or an “immunogenic” epitope or “peptide epitope” is a peptide that comprises an allele-specific motif or supermotif such that the peptide will bind an HLA molecule and induce a CTL and/or HTL response. Thus, immunogenic peptides of the invention are capable of binding to an appropriate HLA molecule and thereafter inducing a cytotoxic T lymphocyte (CTL) response, or a helper T lymphocyte (HTL) response, to the peptide.

The phrases “isolated” or “biologically pure” refer to material which is substantially or essentially free from components which normally accompany the material as it is found in its native state. Thus, isolated peptides in accordance with the invention preferably do not contain materials normally associated with the peptides in their in situ environment. An “isolated” epitope refers to an epitope that does not include the whole sequence of the antigen or polypeptide from which the epitope was derived. Typically the “isolated” epitope does not have attached thereto additional amino acids that result in a sequence that has 100% identity with a native sequence. The native sequence can be a sequence such as a tumor-associated antigen from which the epitope is derived. Thus, the term “isolated” means that the material is removed from its original environment (e.g., the natural environment if it is naturally occurring). For example, a naturally-occurring polynucleotide or peptide present in a living animal is not isolated, but the same polynucleotide or peptide, separated from some or all of the coexisting materials in the natural system, is isolated. Such a polynucleotide could be part of a vector, and/or such a polynucleotide or peptide could be part of a composition, and still be “isolated” in that such vector or composition is not part of its natural environment. Isolated RNA molecules include in vivo or in vitro RNA transcripts of the DNA molecules of the present invention, and further include such molecules produced synthetically.

“Major Histocompatibility Complex” or “MHC” is a cluster of genes that plays a role in control of the cellular interactions responsible for physiologic immune responses. In humans, the MHC complex is also known as the human leukocyte antigen (HLA) complex. For a detailed description of the MHC and HLA complexes, see, Paul, FUNDAMENTAL IMMUNOLOGY, 3^(RD) ED., Raven Press, New York (1993).

The term “motif” refers to a pattern of residues in an amino acid sequence of defined length, preferably a peptide of less than about 15 amino acids in length, or less than about 13 amino acids in length, usually from about 8 to about 13 amino acids (e.g., 8, 9, 10, 11, 12, or 13) for a class I HLA motif and from about 6 to about 25 amino acids (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) for a class II HLA motif, which is recognized by a particular HLA molecule. Motifs are typically different for each HLA protein encoded by a given human HLA allele. These motifs often differ in their pattern of the primary and secondary anchor residues. See Tables 2-4.

A “native” or a “wild type” sequence refers to a sequence found in nature.

A “negative binding residue” or “deleterious residue” is an amino acid which, if present at certain positions (typically not primary anchor positions) in a peptide epitope, results in decreased binding affinity of the peptide for the peptide's corresponding HLA molecule.

The term “peptide” is used interchangeably with “oligopeptide” in the present specification to designate a series of residues, typically L-amino acids, connected one to the other, typically by peptide bonds between the α-amino and carboxyl groups of adjacent amino acids.

A “PanDR binding” peptide or “PADRE®” peptide (Epimmune, San Diego, Calif.) is a member of a family of molecules that binds more than one HLA class II DR molecule. The pattern that defines the PADRE® family of molecules can be referred to as an HLA Class II supermotif. A PADRE® molecule binds to HLA-DR molecules and stimulates in vitro and in vivo human helper T lymphocyte (HTL) responses. For a further definition of the PADRE® family, see copending application U.S. Ser. No. 09/709,774, filed Nov. 11, 2000; and Ser. No. 09/707,738, filed Nov. 6, 2000; PCT publication Nos WO 95/07707, and WO 97/26784; U.S. Pat. No. 5,736,142 issued Apr. 7, 1998; U.S. Pat. No. 5,679,640, issued Oct. 21, 1997; and U.S. Pat. No. 6,413,935, issued Jul. 2, 2002.

“Pharmaceutically acceptable” refers to a generally non-toxic, inert, and/or physiologically compatible composition or component of a composition.

A “pharmaceutical excipient” or “excipient” comprises a material such as an adjuvant, a carrier, pH-adjusting and buffering agents, tonicity adjusting agents, wetting agents, preservatives, and the like. A “pharmaceutical excipient” is an excipient which is pharmaceutically acceptable.

A “primary anchor residue” is an amino acid at a specific position along a peptide sequence which is understood to provide a contact point between the immunogenic peptide and the HLA molecule. One, two or three, primary anchor residues within a peptide of defined length generally defines a “motif” for an immunogenic peptide. These residues are understood to fit in close contact with peptide binding grooves of an HLA molecule, with their side chains buried in specific pockets of the binding grooves themselves. In one embodiment of an HLA class I motif, the primary anchor residues are, located at position 2 (from the amino terminal position) and at the carboxyl terminal position of a peptide epitope in accordance with the invention. The primary anchor positions for each motif and supermotif of HLA Class I are set forth in Table 14. For example, analog peptides can be created by altering the presence or absence of particular residues in these anchor positions. Such analogs are used to modulate the binding affinity of an epitope comprising a particular motif or supermotif.

“Promiscuous recognition” by a TCR is where a distinct peptide is recognized by the various T cell clones in the context of various HLA molecules. Promiscuous binding by an HLA molecule is synonymous with cross-reactive binding.

A “protective immune response” or “therapeutic immune response” refers to a CTL and/or an HTL response to an antigen derived from an pathogenic antigen (e.g., an antigen from an infectious agent or a tumor antigen), which in some way prevents or at least partially arrests disease symptoms, side effects or progression. The immune response may also include an antibody response which has been facilitated by the stimulation of helper T cells.

The term “residue” refers to an amino acid or amino acid mimetic incorporated into a peptide or protein by an amide bond or amide bond mimetic.

A “secondary anchor residue” is an amino acid at a position other than a primary anchor position in a peptide which may influence peptide binding. A secondary anchor residue occurs at a significantly higher frequency amongst HLA-bound peptides than would be expected by random distribution of amino acids at a given position. A secondary anchor residue can be identified as a residue which is present at a higher frequency among high or intermediate affinity binding peptides, or a residue otherwise associated with high or intermediate affinity binding. The secondary anchor residues are said to occur at “secondary anchor positions.” For example, analog peptides can be created by altering the presence or absence of particular residues in these secondary anchor positions. Such analogs are used to finely modulate the binding affinity of an epitope comprising a particular motif or supermotif. The terminology “fixed peptide” is generally used to refer to an analog peptide that has changes in primary anchore position: not secondary.

A “subdominant epitope” is an epitope which evokes little or no response upon immunization with a whole antigen or a fragment of the whole antigen comprising a subdominant epitope and a dominant epitope, which comprise the epitope, but for which a response can be obtained by immunization with an isolated peptide, and this response (unlike the case of cryptic epitopes) is detected when whole antigen or a fragment of the whole antigen comprising a subdominant epitope and a dominant epitope is used to recall the response in vitro or in vivo.

A “supermotif” is a peptide binding specificity shared by HLA molecules encoded by two or more HA alleles. Preferably, a supermotif-bearing peptide is recognized with high or intermediate affinity (as defined herein) by two or more HLA antigens.

“Synthetic peptide” refers to a peptide that is abtained from a non-natural source, e.g., is man-made. Such peptides may be produced using such methods as chemical synthesis or recombinant DNA technology. “Synthetic peptides” include “fusion proteins.”

As used herein, a “vaccine” is a composition used for vaccination, e.g., for prophylaxis or therapy, that comprises one or more peptides of the invention. There are numerous embodiments of vaccines in accordance with the invention, such as by a cocktail of one or more peptides; one or more peptides of the invention comprised by a polyepitopic peptide; or nucleic acids that encode such peptides or polypeptides, e.g., a minigene that encodes a polyepitopic peptide. The “one or more peptides” can include any whole unit integer from 1-150, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, or 150 or more peptides of the invention. The peptides or polypeptides can optionally be modified, such as by lipidation, addition of targeting or other sequences. HLA class I-binding peptides of the invention can be linked to HLA class II-binding peptides, e.g., a PADRE® universal HTL-bindind peptide, to facilitate activation of both cytotoxic T lymphocytes and helper T lymphocytes. Vaccines can comprise peptide pulsed antigen presenting cells, e.g., dendritic cells.

The nomenclature used to describe peptides/proteins follows the conventional practice wherein the amino group is presented to the left (the N-terminus) and the carboxyl group to the right (the C-terminus) of each amino acid residue. When amino acid residue positions are referred to in a peptide epitope they are numbered in an amino to carboxyl direction with position one being the position closest to the amino terminal end of the epitope, or the peptide or protein of which it may be a part. In the formulae representing selected specific embodiments of the present invention, the amino- and carboxyl-terminal groups, although not specifically shown, are in the form they would assume at physiologic pH values, unless otherwise specified. In the amino acid structure formulae, each residue is generally represented by standard three letter or single letter designations. The L-form of an amino acid residue is represented by a capital single letter or a capital first letter of a three-letter symbol, and the D-form for those amino acids having D-forms is represented by a lower case single letter or a lower case three letter symbol. However, when three letter symbols or full names are used without capitals, they may refer to L amino acids. Glycine has no asymmetric carbon atom and is simply referred to as “Gly” or “G”. The amino acid sequences of peptides set forth herein are generally designated using the standard single letter symbol. (A, Alanine; C, Cysteine; D, Aspartic Acid; E, Glutamic Acid; F, Phenylalanine; G, Glycine; H, Histidine; I, Isoleucine; K, Lysine; L, Leucine; M, Methionine; N, Asparagine; P, Proline; Q, Glutamine; R, Arginine; S, Serine; T, Threonine; V, Valine; W, Tryptophan; and Y, Tyrosine.) In addition to these symbols, “B” in the single letter abbreviations used herein designates α-amino butyric acid. In some embodiments, α-amino butyric acid may be replaced with cysteine.

Acronyms used herein are as follows:

-   APC: Antigen presenting cell -   CD3: Pan T cell marker -   CD4: Helper T lymphocyte marker -   CD8: Cytotoxic T lymphocyte marker -   CEA: Carcinoembryonic antigen (see, e.g., SEQ ID NO: 363) -   CTL: Cytotoxic T lymphocyte -   DC: Dendritic cells. DC functioned as potent antigen presenting     cells by stimulating cytokine release from CTL lines that were     specific for a model peptide derived from hepatitis B virus. In vivo     experiments using DC pulsed ex vivo with an HBV peptide epitope have     stimulated CTL immune responses in vivo following delivery to naïve     mice. -   DLT: Dose-limiting toxicity, an adverse event related to therapy. -   DMSO: Dimethylsulfoxide -   ELISA: Enzyme-linked immunosorbant assay -   E:T: Effector:Target ratio -   G-CSF: Granulocyte colony-stimulating factor -   GM-CSF: Granulocyte-macrophage (monocyte)-colony stimulating factor -   HBV: Hepatitis B virus -   HBR2/neu: A tumor associated antigen; cerbB-2 is a synonym (see,     e.g., SEQ ID NO: 364) -   HLA: Human leukocyte antigen -   HLA-DR: Human leukocyte antigen class II -   HPLC: High Performance Liquid Chromatography -   HTC: Helper T Cell -   HTL: Helper T Lymphocyte. A synonym for HTC. -   ID: Identity -   IFNγ: Interferon gamma -   IL-4: Interleukin-4 -   IV: Intravenous -   LU_(30%): Cytotoxic activity for 10⁶ effector cells required to     achieve 30% lysis of a target cell population, at a 100:1 (E:T)     ratio. -   MAb: Monoclonal antibody -   MAGE: Melanoma antigen (see, e.g., SEQ ID NO: 365 and 366 for MAGE2     and MAGE3) -   MLR: Mixed lymphocyte reaction -   MNC: Mononuclear cells -   PB: Peripheral blood -   PBMC: Peripheral blood mononuclear cell -   ProGP™: Progenipoietinl product (Searle, St. Louis, Mo.), a chimeric     flt3/G-CSF receptor agonist. -   SC: Subcutaneous -   S.E.M.: Standard error of the mean -   QD: Once a day dosing -   TAA: Tumor Associated Antigen -   TNF: Tumor necrosis factor -   WBC: White blood cells

The following describes the peptides, corresponding nucleic acid molecules, compositions, and methods of the invention in more detail.

A2, A3, B7, A1, A24 and B44 Peptides and Polynucleotides of Tumor Associated Antigens

A2, A3, B7, A1, A24 and B44 Epitopes and Analogs. In some embodiments, the invention is directed to an isolated peptide comprising or consisting of an epitope and/or analog. In some embodiments, the invention is directed to an isolated polynucleotide encoding such a peptide.

The isolated epitopes and analogs of the invention are all class I binding peptides, i.e., CTL peptides. In particular, the epitopes and analogs of the invention comprise an A2 motif or supermotif, an A3 motif or supermotif, a B7 motif or supermotif, a 144 motif or supermotif, an A1 motif, or an A24 motif. Epitopes and analogs of the invention are those set forth in Tables 6, 9 and 10 (SEQ ID Nos:1-25), 16a-23 (SEQ ID NOs:42-362) and 26-30 (SEQ ID Nos:368-745). Preferred epitopes and analogs are set forth in Tables 10 (SEQ ID Nos:1, 3, 4, 5, 10, 17, 19, 20, 21, and 25) and 20-23 (SEQ ID NOs: 42, 44, 46, 51, 52, 54, 55, 57, 60, 62, 67, 68, 69, 70, 73, 75, 77, 82, 90, 91, 96, 99, 102, 103 104, 107, 111, 114, 116, 119, and 124; 133, 136, 140, 146, 153, 155, and 362; 161, 167, 170, 172, 178, 180, 181, 182, 186, 188, 189, 191, 194, 198, 200, 201, 108, 211, 216, 219, 221, 228, 230, 234, 236, 238, 239, 240, 242, and 246; and 256, 263, 265, 269, 272, 278, 279, 281, 282, 285, 287, 290, 292, 293, 304, 305, 308, 310, 316, 321, 324, 325, 331-336, 344, 345, 351, 356, 361, and 362; respectively). A1, A2, A3, A24, 137 and B44 epitopes and analogs of the invention may be referred to herein as “epitopes” and “analogs” or referred to by Table or referred to by SEQ ID NO. Other epitopes and analogs are referred to herein as CTL epitopes or CTL peptides and HTL epitopes or HTL peptides.

Peptides and Polynucleotides. In some embodiments, the invention is directed to an isolated peptide comprising or consisting of an epitope and/or analog, wherein the epitope or analog consists of a sequence selected from those in tables 6, 9, 10 (SEQ ID Nos:1-25), 16a-23 (SEQ ID NOs:42-362) and 26-30 (SEQ ID Nos:368-745).

Preferably, the peptide comprises or consists of an epitope or analog consisting of a sequence in Tables 10, 20-23, or 26-30.

Peptides of the invention may be fusion proteins of epitope(s) and/or analog(s) to CTL epitope(s), and/or HTL epitope(s), and/or linker(s), and/or spacer(s), and/or carrier(s), and/or additional amino acid(s), and/or may comprise or consist of homopolymers of an epitope or analog or heteropolymers of epitopes and/or analogs, as is described in detail below.

Peptides which comprise an epitope and/or analog of the invention may comprise or consist of a fragment of an antigen (“fragment” or “antigenic fragment”), wherein the fragment comprises an epitope and/or analog. The fragment may be a portion of CEA, HER2/neu MAGE2, MAGE3, and/or p53 (SEQ ID Nos:363-367, respectively). The epitope of the invention may be within the fragment or may be linked directly or indirectly, to the fragment.

The fragment may comprise or consist of a region of a native antigen that contains a high concentration of class I and/or class II epitopes, preferably it contains the greatest number of epitopes per amino acid length. Such epitopes can be present in a frame-shifted manner, e.g. a 10 amino acid long peptide could contain two 9 amino acid long epitopes and one 10 amino acid long epitope.

The fragment may be less than or equal to 600 amino acids, less than or equal to 500 amino acids, less than or equal to 400 amino acids, less than or equal to 250 amino acids, less than or equal to 100 amino acids, less than or equal to 85 amino acids, less than or equal to 75 amino acids, less than or equal to 65 amino acids, or less than or equal to 50 amino acids in length. In certain embodiments, a fragment is less than 101 amino acids in length, in any increment down to 5 amino acids in length. For example, the fragment may be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 amino acids in length. (See Table 33). Fragments of full length antigens may be fragments from about residue 1-20, 21-40, 41-60, 61-80, 81-100, 101-120, 121-140, 141-160, 161-180, 181-200, 201-220, 221-240, 241-260, 261-280, 281-300, 301-320, 321-340, 341-360, 361-380, 381-400, 401-420, 421-440, 441-460, 461-480, 481-500, 501-520, 521-540, 541-560, 561-580, 581-600, 601-620, 621-680, 681-700, 701-720, 721-740, 741-780, 781-800, 801-820, 821-840, 841-860, 861-880, 881-900, 901-920, 921-940, 941-960, 961-980, 981 to the C-terminus of the antigen.

Peptides which comprise an epitope and/or analog of the invention may be a fusion protein comprising one or more amino acid residues in addition to the epitope, analog, or fragment. Fusion proteins include homopolymers and heteropolymers, as described below.

In some embodiments, the peptide comprises or consists of multiple epitopes and/or analogs, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 epitopes and/or analogs of the invention. In other embodiments, the peptide comprises or consists of 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 epitopes and/or analogs of the invention. In some embodiments, the peptide comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25 epitopes and/or analogs of the invention.

For example, the peptide may comprise or consist of at least 1, at least 2, at least 3, at least 4, or all 5 CEA epitopes and/or analogs from Table 6; at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or all 10 HER2/neu epitopes and/or analogs of Table 6; at least 1, at least 2, at least 3, at least 4, or all 5 MAGE2/3 epitopes and/or analogs from Table 6; at least 1, at least 2, at least 3, at least 4, or all 5 p53 epitopes and/or analogs from Table 6. The peptide may comprise or consist of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, or all 14 epitopes and/or analogs from Table 16a; at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or all 20 epitopes and/or analogs from Table 16b; at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, or all 28 epitopes and/or analogs from Table 16c; at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or all 25 epitopes and/or analogs from Table 16d. The peptide may comprise or consist of at least 1, at least 2, at least 3, at least 4, or all 5 epitopes and/or analogs from Table 17a; at least 1, at least 2, at least 3, at least 4, at least 5, or all 6 epitopes and/or analogs from Table 17b; at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, or all 14 epitopes and/or analogs from Table 17c; at least 1, at least 2, at least 3, or all 4 epitopes and/or analogs from Table 17d. The peptide may comprise or consist of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, or all 27 epitopes and/or analogs from Table 18a; at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, or all 24 epitopes and/or analogs from Table 18b; at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, or all 28 epitopes and/or analogs from Table 18c; at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or all 10 epitopes and/or analogs from Table 18d. The peptide may comprise or consist of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, at least 43, at least 44, or all 45 epitopes and/or analogs from Table 19a; at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or all 21 epitopes and/or analogs from Table 19b; at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, or all 40 epitopes and/or analogs from Table 19c; at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, or all 9 epitopes and/or analogs from Table 19d. The peptide may comprise or consist of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, or at least 26 of the epitopes and/or analogs from Table 26, 27, 28, 29, or 30.

The peptide may preferably comprise or consist of at least 1 or all 2 CEA epitopes/analogs of Table 9; at least 1 or all 2 HBER2/neu epitopes/analogs of Table 9; at least 1 or all 2 MAGE2/3 epitopes/analogs of Table 9; at least 1 or all 2 p53 epitopes/analogs of Table 9. The peptide may preferably comprise or consist of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, or all 7 CEA epitopes/analogs of Table 20; at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, or all 9 HER2/neu epitopes/analogs of Table 20; at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, or all 8 MAGE2/3 epitopes/analogs of Table 20; at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, or all 7 p53 epitopes/analogs of Table 20. The peptide may preferably comprise or consist of at least the CEA epitope/analog of Table 21; at least the HER2/neu epitope/analog of Table 21; at least 1, at least 2, at least 3, or all 4 MAGE2/3 epitopes/analogs of Table 21; at least the p53 epitope/analog of Table 21. The peptide may preferably comprise or consist of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, or all 8 CEA epitopes/analogs of Table 22; at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, or all 9 HER2/neu epitopes/analogs of Table 22; at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, or all 8 MAGE2/3 epitopes/analogs of Table 22; at least 1, at least 2, at least 3, at least 4, or all 5 p53 epitopes/analogs of Table 22. The peptide may preferably comprise or consist of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 9, qat least 10, at least 11, or all 12 CEA epitopes/analogs of Table 23; at least 1, at least 2, at least 3, at least 4, at least 5, or all 6 HER2/neu epitopes/analogs of Table 23; at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, or all 13 MAGE2/3 epitopes/analogs of Table 23; at least 1, or all 2 p53 epitopes/analogs of Table 23.

The peptide may comprise or consist of the combinations above and below, and may also exclude any one or several epitopes and/or analogs selected from those in Tables 6, 9, 10 (SEQ ID Nos:1-25), 16a-23 (SEQ ID NOS:42-362) and 26-30 (SEQ ID Nos:368-745). Epitopes/analogs which may preferably be excluded from peptides of the invention are SEQ ID Nos:42, 60, 62, 67, 82, 86, 101, 116, 153, 362, 230, 265, 290, 321, 334, and 345.

The peptide of the invention may comprise or consist of combinations of epitopes and/or analogs including:

A3 CEA combinations such as: (a) SEQ ID NOs:42, 44, 46, 51, 52, 54, and 55; (b) SEQ ID NOs: 44, 46, 51, 52, 54, and 55; (c) SEQ ID NOs:46, 51, 52, 54, and 55; (d) SEQ ID NOs: 51, 52, 54, and 55; (e) SEQ ID NO: 52, 54, and 55; (i) SEQ ID NO: 54 and 55;

(g) SEQ ID NO: 44, 46, 51, 52, and 54; (h) SEQ ID NO: 44, 46, 51, and 52; (i) SEQ ID NO: 44, 46, and 51; and 0) SEQ ID NO: 44 and 46;

(l) SEQ ID NO: 44, 51, 52, 54, and 55; (m) SEQ ID NO: 44, 46, 52, 54, and 55; (n) SEQ ID NO: 44, 46, 51, 54, and 55; and (O) SEQ ID NO: 44, 46, 51, 52, and 55;

A3 HER2/neu combinations such as: (a) SEQ ID NO:57, 60, 62, 67, 68, 69, 70, 73, and 75; (b) SEQ ID NO: 60, 62, 67, 68, 69, 70, 73, and 75; (c) SEQ ID NO: 62, 67, 68, 69, 70, 73, and 75; (d) 67, 68, 69, 70, 73, and 75; (e) 68, 69, 70, 73, and 75; (f) SEQ ID NO: 69, 70, 73, and 75;

(g) SEQ ID NO: 70, 73, and 75; (h) SEQ ID NO: 73 and 75;

(i) SEQ ID NO: 57, 60, 62, 67, 68, 69, 70, and 73; 0) SEQ ID NO: 57, 60, 62, 67, 68, 69, and 70; (k) SEQ ID NO: 57, 60, 62, 67, 68, and 69; (1) SEQ ID NO: 57, 60, 62, 67, and 68;

(m) SEQ ID NO: 57, 60, 62, and 67; (n) SEQ ID NO: 57, 60, and 62; (o) SEQ ID NO: 57 and 60; (p) SEQ ID NO: 57, 68, 69, 70, 73, and 75; (q) SEQ ID NO: 57, 60, 68, 69, 70, 73, and 75; (r) SEQ ID NO: 57, 60, 62, 69, 70, 73, and 75; and (s) SEQ ID NO: 57, 60, 62, 67, 68, 73, and 75;

A3 MAGE2/3 combinations such as: (a) SEQ ID NO:82, 90, 91, 96, 99, 102, and 103; (b) SEQ ID NO: 90, 91, 96, 99, 102, and 103; (c) SEQ ID NO: 91, 96, 99, 102, and 103; (d) SEQ ID NO: 96, 99, 102, and 103; (e) SEQ ID NO: 99, 102, and 103; (f) SEQ ID NO: 102 and 103;

(g) SEQ ID NO: 77, 82, 90, 91, 96, 99, and 102; (h) SEQ ID NO: 77, 82, 90, 91, 96, and 99; (i) SEQ ID NO: 77, 82, 90, 91, and 96; (j) SEQ ID NO: 77, 82, 90, and 91; (k) SEQ ID NO: 77, 82, 90, and 91, 96, 99, 102, and 103; (l) SEQ ID ON: 77, 82, and 90; and (m) SEQ ID NO: 77 and 82;

A3 p53 combinations such as: (a) SEQ ID NO: 107, 111, 114, 116, 119, and 124; (b) SEQ ID NO: 111, 114, 116, 119, and 124; (c) SEQ ID NO: 114, 116, 119, and 124; (d) SEQ ID NO: 116, 119, and 124; (e) SEQ ID NO: 119 and 124;

(f) SEQ ID NO:104, 107, 111, 114, 116, and 119; (g) SEQ ID NO:104, 107, 111, 114, and 116; (h) SEQ ID NO:104, 107, 111, and 114; (i) SEQ ID NO:104, 107, and 111; (j) SEQ ID NO:104 and 107;

(k) SEQ ID NO:104, 111, 114, 116, 119, and 124; (l) SEQ ID NO:104, 107, 114, 116, 119, and 124; (m) SEQ ID NO:104, 107, 111, 116, 119, and 124; (n) SEQ ID NO:104, 107, 111, 114, 116, and 124;

B7 MAGE2/3 combinations such as: (a) SEQ ID NO: 146, 153, and 364; (b) SEQ ID NO: 153 and 364; (d) SEQ ID NO: 140, 146, and 153; (e) SEQ ID NO: 140, 146, and 364;

B7 combinations such as: (a) SEQ ID NO:133, 136, 140, 146, 153, and 155; (b) SEQ ID NO: 136, 140, 146, 153, and 155; (c) SEQ ID NO: 140, 146, 153, and 155; (d) SEQ ID NO: 153 and 155;

A1 CEA combinations such as: (a) SEQ ID NO: 167, 170, 172, 178, 180, 181, and 182; (b) SEQ ID NO: 170, 172, 178, 180, 181, and 182; (c) SEQ ID NO: 172, 178, 180, 181, and 182; (d) SEQ ID NO: 178, 180, 181, and 182; (e) SEQ ID NO:180, 181, and 182; (f) SEQ ID NO: 181 and 182; (g) SEQ ID NO: 161, 167, 170, 172, 178, 180, and 181; (h) SEQ ID NO: 161, 167, 170, 172, 178, and 180; (i) SEQ ID NO: 161, 167, 170, 172, and 178; (j) SEQ ID NO: 161, 167, and 170; (k) SEQ ID NO: 181 and 182;

A1 HER2/neu combinations such as: (a) SEQ ID NO:188, 189, 191, 194, 198, 200, 201, and 208; (b) SEQ ID NO: 189, 191, 194, 198, 200, 201, and 208; (c) SEQ ID NO: 191, 194, 198, 200, 201, and 208; (d) SEQ ID NO: 194, 198, 200, 201, and 208; (e) SEQ ID NO: 198, 200, 201, and 208; (f) SEQ ID NO: 200, 201, and 208; (g) SEQ ID NO:201 and 208;

(h) SEQ ID NO:186, 188, 189, 191, 194, 198, 200, and 201; (i) SEQ ID NO:186, 188, 189, 191, 194, 198, and 200; (j) SEQ ID NO:186, 188, 189, 191, 194, and 198; (k) SEQ ID NO:186, 188, 189, 191, and 194; (l) SEQ ID NO:186, 188, 189, and 191; (m) SEQ ID NO:186, 188, and 189; (n) SEQ ID NO:186 and 188;

A1 MAGE2/3 combinations such as: (a) SEQ ID NO: 216, 219, 221, 228, 230, 234, and 236; (b) SEQ ID NO: 219, 221, 228, 230, 234, and 236; (c) SEQ ED NO: 221, 228, 230, 234, and 236; (d) SEQ ID NO: 228, 230, 234, and 236; (e) SEQ ID NO: 230, 234, and 236; (f) SEQ ID NO: 234 and 236; (g) SEQ ID NO:211, 216, 219, 221, 228, 230, and 234; (h) SEQ ID NO:211, 216, 219, 221, 228, and 230; (i) SEQ ID NO:211, 216, 219, 221, and 228; 6 SEQ ID NO:211, 216, 219, and 221; (k) SEQ ID NO:211, 216, and 219; (l) SEQ ID NO:211 and 216; (m) SEQ ID NO:211, 216, 219, 221, 228, 234, and 236;

A1 p53 combinations such as: (a) SEQ ID NO: 239, 240, 242, and 246; (b) SEQ ID NO: 240, 242, and 246; (c) SEQ ID NO: 242 and 246; (d) SEQ ID NO:238, 239, 240, and 242; (e) SEQ ID NO:238, 239, and 240; (t) SEQ ID NO:238 and 239; (g) SEQ ID NO:238, 240, 242, and 246; (h) SEQ ID NO:238, 239, 242, and 246; (i) SEQ ID NO:238, 239, 240, and 246;

A24 CEA combinations such as: (a) SEQ ID NO: 263, 265, 269, 272, 278, 279, 281, 282, 285, 287, and 290; (b) SEQ ID NO: 265, 269, 272, 278, 279, 281, 282, 285, 287, and 290; (c) SEQ ID NO: 269, 272, 278, 279, 281, 282, 285, 287, and 290; (d) SEQ ID NO: 272, 278, 279, 281, 282, 285, 287, and 290; (e) SEQ ID NO: 278, 279, 281, 282, 285, 287, and 290; (f) SEQ ID NO: 279, 281, 282, 285, 287, and 290; (g) SEQ ID NO: 281, 282, 285, 287, and 290; (h) SEQ ID NO: 282, 285, 287, and 290; (i) SEQ ED NO: 285, 287, and 290; (j) SEQ ID NO: 287 and 290;

(k) SEQ ID NO:256, 263, 265, 269, 272, 278, 279, 281, 282, 285, and 287; (l) SEQ ID NO:256, 263, 265, 269, 272, 278, 279, 281, 282, and 285; (m) SEQ ID NO:256, 263, 265, 269, 272, 278, 279, 281, and 282; (n) SEQ ID NO:256, 263, 265, 269, 272, 278, 279, and 281; (o) SEQ ID NO:256, 263, 265, 269, 272, 278, and 279; (p) SEQ ID NO:256, 263, 265, 269, 272, and 278; (q) SEQ ID NO:256, 263, 265, 269, and 272; (r) SEQ ID NO:256, 263, 265, and 269; (s) SEQ ID NO:256, 263, and 265; (t) SEQ ID NO:256 and 263; (u) SEQ ID NO:256, 263, 269, 272, 278, 279, 281, 282, 285, and 287;

A24 HER2/neu combinations such as: (a) SEQ ID NO: 293, 304, 305, 308, and 310; (b) SEQ ID NO: 304, 305, 308, and 310; (c) SEQ ID NO: 305, 308, and 310; (d) SEQ ID NO: 308 and 310; (e) SEQ ID NO:292, 293, 304, 305, and 308; (f) SEQ ID NO:292, 293, 304, and 305; (g) SEQ ID NO:292, 293, and 304; (h) SEQ ID NO:292 and 293; (i) SEQ ID NO:292, 304, 305, 308, and 310; (j) SEQ ED NO:292, 293, 305, 308, and 310; (k) SEQ ID NO:292, 293, 304, 308, and 310; (l) SEQ ID NO:292, 293, 304, 305, and 310;

A24 MAGE2/3 combinations such as: (a) SEQ ID NO: 321, 324, 325, 331, 332, 333, 334, 335, 336, 344, 345, and 351; (b) SEQ ID NO: 324, 325, 331, 332, 333, 334, 335, 336, 344, 345, and 351; (c) SEQ ID NO: 325, 331, 332, 333, 334, 335, 336, 344, 345, and 351; (d) SEQ ID NO: 331, 332, 333, 334, 335, 336, 344, 345, and 351; (e) SEQ ID NO: 332, 333, 334, 335, 336, 344, 345, and 351; (f) SEQ ID NO: 333, 334, 335, 336, 344, 345, and 351; (g) SEQ ID NO: 333, 334, 335, 336, 344, 345, and 351; (h) SEQ ID NO: 334, 335, 336, 344, 345, and 351; (i) SEQ ID NO: 335, 336, 344, 345, and 351; (j) SEQ ID NO: 336, 344, 345, and 351; (k) SEQ ID NO: 344, 345, and 351; (l) SEQ ID NO:345 and 351;

(m) SEQ ID NO:316, 321, 324, 325, 331, 332, 333, 334, 335, 336, 344, and 345; (n) SEQ ID NO:316, 321, 324, 325, 331, 332, 333, 334, 335, 336, and 344; (o) SEQ ID NO:316, 321, 324, 325, 331, 332, 333, 334, 335, and 336; (p) SEQ ID NO:316, 321, 324, 325, 331, 332, 333, 334, and 335; (q) SEQ ID NO:316, 321, 324, 325, 331, 332, 333, and 334; (r) SEQ ID NO:316, 321, 324, 325, 331, 332, and 333; (s) SEQ ID NO:316, 321, 324, 325, 331, and 332; (t) SEQ ID NO:316, 321, 324, 325, and 331; (u) SEQ ID NO:316, 321, 324, and 325; (v) SEQ ID NO:316, 321, and 324; (w) SEQ ID NO:316 and 321, (x) SEQ ID NO:316, 324, 325, 331, 332, 333, 335, 336, 344, and 351;

A24 p53 combinations such as: SEQ ID NO:356 and 361;

B44 CEA combinations such as: (a) SEQ ID NO:368, 369, 390, 399, and 403; (b) SEQ ID NO:369, 370, 375, 376, 377, and 420; and (c) SEQ ID NO:370, 375, 379, 386, and 429;

B44 HER2/neu combinations such as: (a) SEQ ID NO:432, 435, 436, 443, 448, 460, 466, 467, and 488; (b) SEQ ID NO: 439, 473, 490, and 499; (c) SEQ ID NO:432, 433, 440, 441, 447, 456, 459, and 471; (d) SEQ ID NO: 477, 490, 499, 508, 527, and 535;

B44 MAGE2 combinations such as: (a) SEQ ID NO: 645, 646, 647, 653, 665, 670, 698, 718, and 716; (b) SEQ ID NO: 663, 688, 692, and 701; (c) SEQ ID NO:648, 655, 669, 677, 691, and 700; (d) SEQ ID NO: 651 and 673;

B44 MAGE3 combinations such as: (a) SEQ ID NO: 719, 720, 726, 732, and 740; (b) SEQ ID NO: 721, 725, 726, and 737; (c) SEQ ID NO: 726, 739, and 744; (d) SEQ ID NO: 722, 723, 728 and 735; (e) SEQ ID NO: 720, 728, 731, 736, and 741;

B44 p53 combinations such as: (a) SEQ ID NO: 598, 602, 603, and 617; (b) SEQ ID NO: 589, 599, 600, and 605; (c) SEQ ED NO:600, 603, 604, and 607; (d) SEQ ID NO: 601, 602, 604, and 609;

A2 combinations such as: (a) SEQ ID NO: 6, 8, 16, 18, 22, 23, and 24; (b) SEQ ID NO: 8, 16, 18, 22, 23, and 24; (c) SEQ ID NO: 16, 18, 22, 23, and 24; (d) SEQ ID NO: 18, 22, 23, and 24; (e) SEQ ID NO: 23 and 24; (f) SEQ ID NO: 1, 19, 3, and 4; (g) SEQ ID NO: 2, 6, 8, 16, 18, 22, and 23; (h) SEQ ID NO: 2, 6, 8, 16, 18, and 22; (i) SEQ ID NO: 2, 6, 8, 16, 18, and 22; (j) SEQ ID NO: 2, 6, 8, 16, and 18; (k) SEQ ID NO: 2, 6, 8, and 16; (l) SEQ ID NO: 2, 6, and 8; and (m) SEQ ID NO: 2 and 6; (n) SEQ ID NO:3, 4, 5, and 17; (o) SEQ ID NO: 20, 21, and 25; (p) SEQ ID NO: 1, 10, 17, and 25; (q) SEQ ID NO: 4, 5, 10, 17, and 25;

TAA combinations such as: (a) SEQ ID NO: 1, 17, 22, 104, 114, 133, 136, 146, 170, 189, 221, 310, 336, 361, and 399; (b) SEQ ID NO:111, 124, 133, 140, 155, 180, 194, 228, 246, and 281; (c) SEQ ID NO: 16, 18, 25, 43, 68, 117, 309, and 499; (d) SEQ ID NO: 48, 55, 97, 369, 409, and 512; (e) SEQ ID NO: 55, 99, 135, 238, and 602; (f) SEQ ID NO: 1, 58, 77, 104, 128, 166, 207, 240, 360, and 403; (g) SEQ ID NO:17, 50, 72, 130, 161, 199, 300, and 627; (h) SEQ ID NO: 10, 55, 82, 104, 198, 400, 433, and 501; (i) SEQ ID NO: 3, 22, 122, 196, 211, 301, 360, and 667; (j) SEQ ID NO: 1, 21, 44, 100, 207, 405, and 661;

Peptides of the invention may also comprise or consist of combinations of the above combinations, including:

A24 combinations such as: A24 CEA (a) and A24 HER2/neu (a); A24 CEA (a) and A24 MAGE2/3 (a); A24 CEA (a) and A24 p53; A24 CEA (c) and A24 HER2/neu (e); A24 CEA (i) and A24 MAGE2/3 (a); A24 CEA (n) and A24 p53 (k);

A3 combinations such as: A3 CEA (a) and A3 HER2/neu (a); A3 CEA (a) and A3 MAGE2/3 (a); A3 CEA (a) and A3 p53 (a); A3 CEA (d) and A3 HER2/neu (b); A3 CEA (f) and A3 MAGE2/3 (i); A3 CEA (e) and A3 p53 (a);

CEA combinations such as: A24 CEA (a) and A1 CEA (a); A24 CEA (b) and A1 CEA (a); A24 CEA (c) and A1 CEA (a); A24 CEA (c) and A1 CEA (a); A3 CEA (a) and A1 CEA (a); A3 CEA (b) and A1 CEA (a); A3 CEA (c) and A24 CEA (a); B7 CEA (c) and A1 CEA (a);

B7 CEA (a) and A3 CEA (a); B44 CEA (b) and A1 CEA (a); A3 CEA (e) and A1 CEA (g); A3 CEA (i) and A1 CEA (m);

A1 CEA (a), (b) (c), (d), (e), (f) (g), (h), (i), G), or (k) epitopes/analogs, and A3 CEA (a), (b) (c), (d), (e), (f) (g), h), (i), (j), or (k) epitopes/analogs, and B7 CEA (a), (b) (c), (d), (e), (f) (g), (h), (i), (j), or (k) epitopes/analogs, and A3 p53 (a), (b) (c), (d), (e), (f) (g), (h), (i), (j), or (k) epitopes/analogs, and B44 MAGE2 (a), (b) (c), (d), (e), (f) (g), (h), (i), (j), or (k) epitopes/analogs, and A3 MAGE2 (a), (b) (c), (d), (e), (f) (g), (h), (i), (j), or (k) epitopes/analogs;

A24 CEA (a), (b) (c), (d), (e), (f) (g), (h), (i), (j), or (k) epitopes/analogs, and A2 CEA (a), (b) (c), (d), (e), (f) (g), (h), (i), (j), or (k) epitopes/analogs, and B7 MAGE3 (a), (b) (c), (d), (e), (t) (g), (h), (i), (j), or (k) epitopes/analogs, and B44 p53 (a), (b) (c), (d), (e), (f) (g), (h), (i), (j), or (k) epitopes/analogs;

A3 CEA (a), (b) (c), (d), (e), (f) (g), (h), (i), (j), or (k) epitopes/analogs, and B7 p53 (a), (b) (c), (d), (e), (f) (g), (h), (i), (j), or (k) epitopes/analogs, and B44 MAGE3 (a), (b) (c), (d), (e), (f) (g), (h), (i), (j), or (k) epitopes/analogs, and A24 HER2/neu (a), (b) (c), (d), (e), (f) (g), (h), (i), (j), or (k) epitopes/analogs.

The peptide may also comprise or consist of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, or at least 8 peptides selected from the group consisting of the combinations set forth above.

The peptide may also be a homopolymer of one epitope or analog or the peptide may be a heteropolymer which contains at least two different epitopes and/or analogs. Polymers have the advantage of increased probability for immunological reaction and, where different epitopes/analogs are used to make up the polymer, the ability to induce antibodies and/or T cells that react with different antigenic determinants of the antigen(s) targeted for an immune response.

A homopolymer may comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, or 150 copies of the same epitope or analog.

A heteropolymer may comprise one or more copies of an individual epitope or analog and one or more copies of one or more different epitopes and/or analogs of the invention. The epitopes and/or analogs that form a heteropolymer may all be from the same antigen, e.g., may be from CEA, p53, MAGE2/3, HER2/neu or other antigens herein or known in the art, or may be from different antigens, preferably TAAs. Combinations of epitopes and/or analogs that may form a heteropolymer include those combinations described above. Heteropolymers may contain multiple copies of one or more epitopes and/or analogs.

Thus, peptides of the invention such as heteropolymers may comprise a first epitope and/or analog and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 other (different).

Peptides of the invention may also comprise additional amino acids.

In some embodiments, the peptides may comprise a number of CTL and/or HTL epitopes, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 CTL and/or HTL epitopes.

The CTL and/or HTL epitope and the epitope/analog of the invention may be from the same TAA or from different TAAs. Thus, for example, if the epitope and/or analog is from CEA, the CTL peptide and/or HTL peptide may also be from CEA. Alternatively, the CTL peptide and/or HTL peptide may be from another antigen, preferably a TAA antigen such as p53, MAGE2/3 or HER2/neu. As another example, if the epitope and/or analog is from p53, the CTL peptide and/or HTL peptide may be from p53 or, alternatively, may be from MAGE2/3, HER2/neu, or CEA.

The CTL peptide and/or HTL peptide may be from tumor-associated antigens such as but not limited to, melanoma antigens MAGE-1, MAGE-2, MAGE-3, MAGE-11, MAGE-A10, as well as BAGE, GAGE, RAGE, MAGE-C1, LAGE-1, CAG-3, DAM, MUC1, MUC2, MUC18, NY-ESO-1, MUM-1, CDK4, BRCA2, NY-LU-1, NY-LU-7, NY-LU-12, CASP8, RAS, KIAA-2-5, SCCs, p53, p73, CEA, HER2/neu, Melan-A, gp100, tyrosinase, TRP2, gp75/TRP1, kallikrein, prostate-specific membrane antigen (PSM), prostatic acid phosphatase (PAP), prostate-specific antigen (PSA), PT1-1, ∃-catenin, PRAME, Telomerase, FAK, cyclin D1 protein, NOEY2, EGF-R, SART-1, CAPB, HPVE7, p15, Folate receptor CDC27, PAGE-1, and PAGE-4.

Alternatively, the CTL peptide and/or HTL peptide may be from other antigens including hepatitis B core and surface antigens (HBVc, HBVs), hepatitis C antigens, Epstein-Barr virus antigens, human immunodeficiency virus (HIV) antigens and human papiroma virus (HPV) antigens (in particular anitgens from HPV-16, HPV-18, HPV-31, HPV-33, HPV-45, HPV-52, HPV-56 and HPV-58, Mycobacterium tuberculosis and Chlamydia. Examples of suitable fungal antigens include those derived from Candida albicans, Cryptococcus neoformans, Coccidoides spp., Histoplasma spp, and Aspergillus fumigatis. Examples of suitable protozoan parasitic antigens include those derived from Plasmodium spp., including P. falciparum, Trypanosoma spp., Schistosoma spp., Leishmania spp and the like.

A CTL epitope may comprise a sequence selected from the group consisting of: SEQ ID Nos:1-25.

Examples of CTL peptides and HTL peptides are disclosed in WO 01/42270, published 14 Jun. 2001; WO 01/41788, publidhes 14 Jun. 2001; WO 01/42270, published 14 Jun. 2001; WO 01/45728, published 28 Jun. 2001; and WO 01/41787, published 14 Jun. 2001.

The HTL peptide may comprise a “loosely HLA-restricted” or “promiscuous” sequence. Examples of amino acid sequences that are promiscuous include sequences from antigens such as tetanus toxoid at positions 830-843 (QYIKANSKFIGITE; SEQ ID NO: 627), Plasmodium falciparum Cs protein at positions 378-398 (DIEKKIAKMKASSVFNVVNS; SEQ ID NO: 628), and Streptococcus 18 kD protein at positions 116-131 (GAVDSILGGVATYGAA; SEQ ID NO: 629). Other examples include peptides bearing a DR 1-4-7 supermotif, or either of the DR3 motifs.

The HTL peptide may comprise a synthetic peptide such as a Pan-DR-binding epitope (e.g., a PADRE® peptide, Epimmune Inc., San Diego, Calif., described, for example, in U.S. Pat. No. 5,736,142), for example, having the formula aKXVAAZTLKAAa, where “X” is either cyclohexylalanine, phenylalanine, or tyrosine; “Z” is either tryptophan, tyrosine, histidine or asparagine; and “a” is either D-alanine or L-alanine (SEQ ID NO: 746). Certain pan-DR binding epitopes comprise all “L” natural amino acids; these molecules can be provided as peptides or in the form of nucleic acids that encode the peptide. See also, U.S. Pat. Nos. 5,679,640 and 6,413,935.

The peptide may comprise additional amino acids. Such additional amino acids may be Ala, Arg, Asn, Asp, Cys, Gln, Gly, Glu, His, Ile, Leu, 35, Lys, Met, Phe, Pro, Ser, Thr, Tyr, Trp, Val, amino acid mimetics, and other unnatural amino acids such as those described below. Additional amino acids may provide for ease of linking peptides one to another, for linking epitopes and/or analogs to one another, for linking epitopes and/or analogs to CTL and/or HTL epitopes, for coupling to a carrier support or larger peptide, for modifying the physical or chemical properties of the peptide or oligopeptide, or the like. Amino acids such as Ala, Arg, Asn, Asp, Cys, Gln, Gly, Glu, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Tyr, Trp, or Val, or the like, can be introduced at the C- and/or N-terminus of the peptide and/or can be introduced internally.

The peptide may comprise an amino acid spacer, which may be joined to the epitopes, analogs, CTL epitopes, HTL epitopes, carriers, etc. within a peptide or may be joined to the peptide at the N-and/or C-terminus. Thus, spacers may be at the N-terminus or C-terminus of peptide, or may be internal such that they link or join epitopes, analogs, CTL epitopes, HTL epitopes, carriers, additional amino acids, and/or antigenic fragments one to the other.

The spacer is typically comprised of one or more relatively small, neutral molecules, such as amino acids or amino acid mimetics, which are substantially uncharged under physiological conditions. The spacers are typically selected from, e.g., Ala, Gly, or other neutral spacers of nonpolar amino acids or neutral polar amino acids. It will be understood that the optionally present spacer may be composed of the same residues or may be composed of one or more different residues and thus may be a homo- or hetero-oligomer of spacer residues. Thus, the spacer may contain more than one Ala residue (poly-alanine) or more than one Gly residue (poly-glycine), or may contain both Ala and Gly residues, e.g., Gly, Gly-Gly-, Ser,Ser-Ser-, Gly-Ser-, Ser-Gly-, etc. When present, the spacer will usually be at least one or two residues, more usually three to six residues and sometimes 10 or more residues, e.g., 3, 4, 5, 6, 7, 8, 9, or 10, or even more residues. (Livingston, B. D. et al. Vaccine 19:4652-4660 (2000)).

Peptides may comprise carriers such as those well known in the art, e.g., thyroglobulin, albumins such as human serum albumin, tetanus toxoid, polyamino acids such as poly L-lysine, poly L-glutamic acid, influenza virus proteins, hepatitis B virus core protein, and the like. (See Table 31).

In addition, the peptide may be modified by terminal-NH₂ acylation, e.g., by alkanoyl (C₁-C₂₀) or thioglycolyl acetylation, terminal-carboxyl amidation, e.g., ammonia, methylamine, etc. In some instances these modifications may provide sites for linking to a support or other molecule.

The peptides in accordance with the invention can contain modifications such as but not limited to glycosylation, side chain oxidation, biotinylation, phosphorylation, addition of a surface active material, e.g. a lipid, or can be chemically modified, e.g., acetylation, etc. Moreover, bonds in the peptide can be other than peptide bonds, e.g., covalent bonds, ester or ether bonds, disulfide bonds, hydrogen bonds, ionic bonds, etc.

Peptides of the present invention may contain substitutions to modify a physical property (e.g., stability or solubility) of the resulting peptide. For example, peptides may be modified by the substitution of a cysteine (C) with α-amino butyric acid (“B”). Due to its chemical nature, cysteine has the propensity to form disulfide bridges and sufficiently alter the peptide structurally so as to reduce binding capacity. Substituting α-amino butyric acid for C not only alleviates this problem, but actually improves binding and crossbinding capability in certain instances. Substitution of cysteine with α-amino butyric acid may occur at any residue of a peptide, e.g., at either anchor or non-anchor positions of an epitope or analog within a peptide, or at other positions of a peptide.

The peptides can comprise amino acid mimetics or unnatural amino acids, e.g. D- or L-naphylalanine; D- or L-phenylglycine; D- or L-2-thieneylalanine; D- or L-1, -2,3-, or 4-pyreneylalanine; D- or L-3 thieneylalanine; D- or L-(2-pyridinyl)-alanine; D- or L-(3-pyridinyl)-alanine; D- or L-(2-pyrazinyl)-alanine; D- or L-(4-isopropyl)-phenylglycine; D-(trifluoromethyl)-phenylglycine; D-(trifluoromethyl)-phenylalanine; D-ρ-fluorophenylalanine; D- or L-ρ-biphenylphenylalanine; D- or L-ρ-methoxybiphenylphenylalanine; D or L-2-indole(alkyl)alanines; and, D- or L-alkylalanines, where the alkyl group can be a substituted or unsubstituted methyl, ethyl, propyl, hexyl, butyl, pentyl, isopropyl, iso-butyl, sec-isotyl, iso-pentyl, or a non-acidic amino acids. Aromatic rings of a non-natural amino acid include, e.g., thiazolyl, thiophenyl, pyrazolyl, benzimidazolyl, naphthyl, furanyl, pyrrolyl, and pyridyl aromatic rings. Modified peptides that have various amino acid mimetics or unnatural amino acids are particularly useful, as they tend to manifest increased stability in vivo. Such peptides may also possess improved shelf-life or manufacturing properties.

Peptide stability can be assayed in a number of ways. For instance, peptidases and various biological media, such as human plasma and serum, have been used to test stability. See, e.g., Verhoef, et al., Eur. J. Drug Metab. Pharmacokinetics 11:291 (1986). Half-life of the peptides of the present invention is conveniently determined using a 25% human serum (v/v) assay. The protocol is generally as follows: Pooled human serum (Type AB, non-heat inactivated) is delipidated by centrifugation before use. The serum is then diluted to 25% with RPMI-1640 or another suitable tissue culture medium. At predetermined time intervals, a small amount of reaction solution is removed and added to either 6% aqueous trichloroacetic acid (TCA) or ethanol. The cloudy reaction sample is cooled (4° C.) for 15 minutes and then spun to pellet the precipitated serum proteins. The presence of the peptides is then determined by reversed-phase HPLC using stability-specific chromatography conditions.

The peptides in accordance with the invention can be a variety of lengths, and either in their neutral (uncharged) forms or in forms which are salts. The peptides in accordance with the invention can contain modifications such as glycosylation, side chain oxidation, or phosphorylation, generally subject to the condition that modifications do not destroy the biological activity of the peptides.

The peptides of the invention may be lyophylized, or may be in crystal form.

It is generally preferable that the epitope be as small as possible while still maintaining substantially all of the immunologic activity of the native protein. When possible, it may be desirable to optimize HLA class I binding epitopes of the invention to a length of about 8 to about 13 amino acid residues, for example, 8, 9, 10, 11, 12 or 13, preferably 9 to 10. It is to be appreciated that one or more epitopes in this size range can be comprised by a longer peptide (see the Definition Section for the term “epitope” for further discussion of peptide length). HLA class II binding epitopes are preferably optimized to a length of about 6 to about 30 amino acids in length, e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30, preferably to between about 13 and about 20 residues, e.g., 13, 14, 15, 16, 17, 18, 19 or 20. Preferably, the epitopes are commensurate in size with endogenously processed pathogen-derived peptides or tumor cell peptides that are bound to the relevant HLA molecules. The identification and preparation of peptides of various lengths can be carried out using the techniques described herein.

Peptides in accordance with the invention can be prepared synthetically, by recombinant DNA technology or chemical synthesis, or can be isolated from natural sources such as native tumors or pathogenic organisms. Epitopes may be synthesized individually or joined directly or indirectly in a peptide. Although the peptide will preferably be substantially free of other naturally occurring host cell proteins and fragments thereof, in some embodiments the peptides may be synthetically conjugated to be joined to native fragments or particles.

The peptides of the invention can be prepared in a wide variety of ways. For relatively short sizes, the peptides can be synthesized in solution or on a solid support in accordance with conventional techniques. Various automatic synthesizers are commercially available and can be used in accordance with known protocols. (See, for example, Stewart & Young, SOLID PHASE PEPTIDE SYNTIHESIS, 2D. ED., Pierce Chemical Co., 1984). Further, individual peptides can be joined using chemical ligation to produce larger peptides that are still within the bounds of the invention.

Alternatively, recombinant DNA technology can be employed wherein a nucleotide sequence which encodes a peptide inserted into an expression vector, transformed or transfected into an appropriate host cell and cultivated under conditions suitable for expression. These procedures are generally known in the art, as described generally in Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989). Thus, recombinant peptides, which comprise or consist of one or more epitopes of the invention, can be used to present the appropriate T cell epitope.

Polynucleotides encoding each of the peptides above are also part of the invention. As appreciated by one of ordinary skill in the art, various nucleic acids will encode the same peptide due to the redundancy of the genetic code. Each of these nucleic acids falls within the scope of the present invention. This embodiment of the invention comprises DNA and RNA, and in certain embodiments a combination of DNA and RNA. It is to be appreciated that any polynucleotide that encodes a peptide in accordance with the invention falls within the scope of this invention.

The polynucleotides encoding peptides contemplated herein can be synthesized by chemical techniques, for example, the phosphotriester method of Matteucci, et al., J. Am. Chem. Soc. 103:3185 (1981). Polynucleotides encoding peptides comprising or consisting of an analog can be made simply by substituting the appropriate and desired nucleic acid base(s) for those that encode the native epitope.

The polynucleotide, e.g. minigene (see below), may be produced by assembling oligonucleotides that encode the plus and minus strands of the polynucleotide, e.g. minigene. Overlapping oligonucleotides (15-100 bases long) may be synthesized, phosphorylated, purified and annealed under appropriate conditions using well known techniques. The ends of the oligonucleotides can be joined, for example, using T4 DNA ligase. A polynucleotide, e.g. minigene, encoding the peptide of the invention, can be cloned into a desired vector such as an expression vector. The coding sequence can then be provided with appropriate linkers and ligated into expression vectors commonly available in the art, and the vectors used to transform suitable hosts to produce the desired peptide such as a fusion protein.

A large number of such vectors and suitable host systems are known to those of skill in the art, and are commercially available. The following vectors are provided by way of example. Bacterial: pQE70, pQE60, pQE-9 (Qiagen), pBS, pD10, phagescript, psiX174, pBluescript SK, pbsks, pNH8A, pNH16a, pNH18A, pNH46A (Stratagene); ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia); pCR (Livitrogen). Eukaryotic: pWLNEO, pSV2CAT, pOG44, pXT1, pSG (Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia); p75.6 (valentis); pCEP (Invitrogen); pCEI (Epimmune). However, any other plasmid or vector can be used as long as it is replicable and viable in the host.

As representative examples of appropriate hosts, there can be mentioned: bacterial cells, such as E. coli, Bacillus subtilis, Salmonella typhimurium and various species within the genera Pseudomonas, Streptomyces, and Staphylococcus; fungal cells, such as yeast; insect cells such as Drosophila and Sf9; animal cells such as COS-7 lines of monkey kidney fibroblasts, described by Gluzman, Cell 23:175 (1981), and other cell lines capable of expressing a compatible vector, for example, the C127, 3T3, CHO, HeLa and BHK cell lines or Bowes melanoma; plant cells, etc. The selection of an appropriate host is deemed to be within the scope of those skilled in the art from the teachings herein.

Thus, the present invention is also directed to vectors, preferably expression vectors useful for the production of the peptides of the present invention, and to host cells comprising such vectors.

Host cells are genetically engineered (transduced or transformed or transfected) with the vectors of this invention which can be, for example, a cloning vector or an expression vector. The vector can be, for example, in the form of a plasmid, a viral particle, a phage, etc. The engineered host cells can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants or amplifying the polynucletides. The culture conditions, such as temperature, pH and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.

For expression of the peptides, the coding sequence will be provided with operably linked start and stop codons, promoter and terminator regions and usually a replication system to provide an expression vector for expression in the desired cellular host. For example, promoter sequences compatible with bacterial hosts are provided in plasmids containing convenient restriction sites for insertion of the desired coding sequence. The resulting expression vectors are transformed into suitable bacterial hosts.

Generally, recombinant expression vectors will include origins of replication and selectable markers permitting transformation of the host cell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiae TRP1 gene, and a promoter derived from a highly-expressed gene to direct transcription of a downstream structural sequence. Such promoters can be derived from operons encoding glycolytic enzymes such as 3-phosphoglycerate kinase (PGK), ∀-factor, acid phosphatase, or heat shock proteins, among others. The heterologous structural sequence is assembled in appropriate phase with translation initiation and termination sequences, and preferably, a leader sequence capable of directing secretion of translated protein into the periplasmic space or extracellular medium. Optionally, the heterologous sequence can encode a fusion protein including an N-terminal identification peptide imparting desired characteristics, e.g., stabilization or simplified purification of expressed recombinant product.

Yeast, insect or mammalian cell hosts may also be used, employing suitable vectors and control sequences. Examples of mammalian expression systems include the COS-7 lines of monkey kidney fibroblasts, described by Gluzman, Cell 23:175 (1981), and other cell lines capable of expressing a compatible vector, for example, the C127, 3T3, CHO, HeLa and BHK cell lines. Mammalian expression vectors will comprise an origin of replication, a suitable promoter and enhancer, and also any necessary ribosome binding sites, polyadenylation site, splice donor and acceptor sites, transcriptional termination sequences, and 5′ flanking nontranscribed sequences. Such promoters may also be derived from viral sources, such as, e.g., human cytomegalovirus (CMV-IE promoter) or herpes simplex virus type-1 (HSV TK promoter). Nucleic acid sequences derived from the SV40 splice, and polyadenylation sites can be used to provide the required nontranscribed genetic elements.

Polynucleotides encoding peptides of the invention may also comprise a ubiquitination signal sequence, and/or a targeting sequence such as an endoplasmic reticulum (ER) signal sequence to facilitate movement of the resulting peptide into the endoplasmic reticulum.

Polynucleotides of the invention, e.g., minigenes, may be expressed in human cells. A human codon usage table can be used to guide the codon choice for each amino acid. Such polynucleotides preferably comprise spacer amino acid residues between epitopes and/or analogs, such as those described above, or may comprise naturally-occurring flanking sequences adjacent to the epitopes and/or analogs (and/or CTL and HTL epitopes).

The peptides of the invention can also be expressed by viral or bacterial vectors. Examples of expression vectors include attenuated viral hosts, such as vaccinia or fowlpox. As an example of this approach, vaccinia virus is used as a vector to express nucleotide sequences that encode the peptides of the invention. Vaccinia vectors and methods useful in immunization protocols are described in, e.g., U.S. Pat. No. 4,722,848. Another vector is BCG (Bacille Calmette Guerin). BCG vectors are described in Stover et al., Nature 351:456-460 (1991). A wide variety of other vectors useful for therapeutic administration or immunization of the polypeptides of the invention, e.g. adeno and adeno-associated virus vectors, retroviral vectors, Salmonella typhi vectors, detoxified anthrax toxin vectors, and the like, will be apparent to those skilled in the art from the description herein. A preferred vector is Modified Vaccinia Ankara (MVA) (e.g., Bavarian Noridic (MVA-BN)).

Standard regulatory sequences well known to those of skill in the art are preferably included in the vector to ensure expression in the human target cells. Several vector elements are desirable: a promoter with a downstream cloning site for polynucleotide, e.g., minigene insertion; a polyadenylation signal for efficient transcription termination; an E. coli origin of replication; and an E. coli selectable marker (e.g. ampicillin or kanamycin resistance). Numerous promoters can be used for this purpose, e.g., the human cytomegalovirus (hCMV) promoter. See, e.g., U.S. Pat. Nos. 5,580,859 and 5,589,466 for other suitable promoter sequences. A preferred promoter is the CMV-IE promoter.

Polynucleotides, e.g. minigenes, may comprise one or more synthetic or naturally-occurring introns in the transcribed region. The inclusion of mRNA stabilization sequences and sequences for replication in mammalian cells may also be considered for increasing polynucleotide, e.g. minigene, expression.

In addition, the polynucleotide, e.g. minigene, may comprise immunostimulatory sequences (ISSs or CpGs). These sequences may be included in the vector, outside the polynucleotide (e.g. minigene) coding sequence to enhance immunogenicity.

In some embodiments, a bi-cistronic expression vector which allows production of both the polynucleotide- (e.g. minigene-) encoded peptides of the invention and a second protein (e.g., one that modulates immunogenicity) can be used. Examples of proteins or polypeptides that, if co-expressed with peptides of the invention, can enhance an immune response include cytokines (e.g., IL-2, IL-12, GM-CSF), cytokine-inducing molecules (e.g., LeIF), costimulatory molecules, or pan-DR binding proteins (PADRE® molecules, Epimmune, San Diego, Calif.). Helper T cell (HTL) epitopes such as PADRE® molecules can be joined to intracellular targeting signals and expressed separately from expressed peptides of the invention. Specifically decreasing the immune response by co-expression of immunosuppressive molecules (e.g. TGF-β) may be beneficial in certain diseases.

Once an expression vector is selected, the polynucleotide, e.g. minigene, is cloned into the polylinker region downstream of the promoter. This plasmid is transformed into an appropriate bacterial strain, and DNA is prepared using standard techniques. The orientation and DNA sequence of the polynucleotide, e.g. minigene, as well as all other elements included in the vector, are confirmed using restriction mapping, DNA sequence analysis, and/or PCR analysis. Bacterial cells harboring the correct plasmid can be stored as cell banks.

Therapeutic/prophylactic quantities of DNA can be produced for example, by fermentation in E. coli, followed by purification. Aliquots from the working cell bank are used to inoculate growth medium, and are grown to saturation in shaker flasks or a bioreactor according to well known techniques. Plasmid DNA is purified using standard bioseparation technologies such as solid phase anion-exchange resins available, e.g., from QIAGEN, Inc. (Valencia, Calif.). If required, supercoiled DNA can be isolated from the open circular and linear forms using gel electrophoresis or other methods.

Purified polynucleotides, e.g. minigenes, can be prepared for injection using a variety of formulations. The simplest of these is reconstitution of lyophilized polynucleotide, e.g. DNA, in sterile phosphate-buffer saline (PBS). This approach, known as “naked DNA,” is currently being used for intramuscular (LM) administration in clinical trials. To maximize the immunotherapeutic effects of polynucleotide vaccines, alternative methods of formulating purified plasmid DNA may be used. A variety of such methods have been described, and new techniques may become available. Cationic lipids, glycolipids, and fusogenic liposomes can also be used in the formulation (see, e.g., WO 93/24640; Mannino & Gould-Fogerite, BioTechniques 6(7): 682 (1988); U.S. Pat. No. 5,279,833; WO 91/06309; and Felgner, et al., Proc. Nat'l Acad. Sci. USA 84:7413 (1987). In addition, peptides and compounds referred to collectively as protective, interactive, non-condensing compounds (PINC) can also be complexed to purified plasmid DNA to influence variables such as stability, intramuscular dispersion, or trafficking to specific organs or cell types.

Known methods in the art can be used to enhance delivery and uptake of a polynucleotide in vivo. For example, the polynucleotide can be complexed to polyvinylpyrrolidone (PVP), to prolong the localized bioavailability of the polynucleotide, thereby enhancing uptake of the polynucleotide by the organisum (see e.g., U.S. Pat. No. 6,040,295; EP 0 465 529; WO 98/17814). PVP is a polyamide that is known to form complexes with a wide variety of substances, and is chemically and physiologically inert.

Target cell sensitization can be used as a functional assay of the expression and HLA class I presentation of polynucleotide- (e.g. minigene-) encoded peptides. For example, the polynucleotide, e.g. plasmid DNA, is introduced into a mammalian cell line that is a suitable target for standard CTL chromium release assays. The transfection method used will be dependent on the final formulation. For example, electroporation can be used for “naked” DNA, whereas cationic lipids or PVP-formulated DNA allow direct in vitro transfection. A plasmid expressing green fluorescent protein (GFP) can be co-transfected to allow enrichment of transfected cells using fluorescence activated cell sorting (FACS). The transfected cells are then chromium-51 (⁵¹Cr) labeled and used as targets for epitope-specific CTLs. Cytolysis of the target cells, detected by ⁵¹Cr release, indicates both production and HLA presentation of, polynucleotide-, e.g. minigene-, encoded epitopes and/or analogs of the invention, or peptides comprising them. Expression of HTL epitopes may be evaluated in an analogous manner using assays to assess HTL activity.

In vivo immunogenicity is a second approach for functional testing of polynucleotides, e.g. minigenes. Transgenic mice expressing appropriate human HLA proteins are immunized with the polynucleotide, e.g. DNA, product. The dose and route of administration are formulation dependent (e.g., IM for polynucleotide (e.g., naked DNA or PVP-formulated DNA) in PBS, intraperitoneal (IP) for lipid-complexed polynucleotide (e.g., DNA)). Eleven to twenty-one days after immunization, splenocytes are harvested and restimulated for one week in the presence of polynucleotides encoding each peptide being tested. Thereafter, for peptides comprising or consisting of epitopes and/or analogs, standard assays are conducted to determine if there is cytolysis of peptide-loaded, ⁵¹Cr-labeled target cells. Once again, lysis of target cells that were exposed to epitopes and/or analogs corresponding to those encoded by the polynucleotide, e.g. minigene, demonstrates polynucleotide, e.g., DNA, vaccine function and induction of CTLs. Immunogenicity of HTL epitopes is evaluated in transgenic mice in an analogous manner.

Alternatively, the nucleic acids can be administered using ballistic delivery as described, for instance, in U.S. Pat. No. 5,204,253. Using this technique, particles comprised solely of a polynucleotide such as DNA are administered. In a further alternative embodiment for ballistic delivery, polynucleotides such as DNA can be adhered to particles, such as gold particles.

The use of polynucleotides such as multi-epitope minigenes is described herein and in, e.g. co-pending application U.S. Ser. No. 09/311,784; Ishioka et al., J. Immunol. 162:3915-3925, 1999; An, L. and Whitton, J. L., J. Virol. 71:2292, 1997; Thomson, S. A. et al., J. Immunol. 157:822, 1996; Whitton, J. L. et al., J. Virol. 67:348, 1993; Hanke, R. et al., Vaccine 16:426, 1998. For example, a polynucleotide such as a multi-epitope DNA plasmid can be engineered which encodes an epitope derived from multiple regions of a TAA (e.g., p53, HER2/nev, MAGE-2/3, or CEA), a pan-DR binding peptide such as the PADRE® universal helper T cell epitope, and an endoplasmic reticulum-translocating signal sequence. As described in the sections above, a peptide/polynucleotide may also comprise/encode epitopes that are derived from other TAAs.

Thus, the invention includes peptides as described herein, polynucleotides encoding each of said peptides, as well as compositions comprising the peptides and polynucleotides, and includes methods for, producing and methods of using the peptides, polynucleotides, and compositions, as further described below.

Compositions. In other embodiments, the invention is directed to a composition comprising one or more peptides and/or a polynucleotide of the invention and optionally another component(s).

In some embodiments, the composition comprises or consists of multiple peptides, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 peptides of the invention. In other embodiments, the composition comprises or consists of 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145 or 150 or more peptides of the invention. In some embodiments, the composition comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, at least 43, at least 44, at least 44, at least 45, at least 46, at least 47, at least 48, at least 49, at least 50, peptides of the invention.

For example, compositions may comprise or consist of combinations of epitopes and/or analogs, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 epitopes and/or analogs of the invention. In other embodiments, the composition comprises or consists of 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 epitopes and/or analogs of the invention. In some embodiments, the composition comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25 epitopes and/or analogs of the invention.

For example, the composition may comprise or consist of at least 1, at least 2, at least 3, at least 4, or all 5 CEA epitopes and/or analogs from Table 6; at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or all 10 HER2/neu epitopes and/or analogs of Table 6; at least 1, at least 2, at least 3, at least 4, or all 5 MAGE2/3 epitopes and/or analogs from Table 6; at least 1, at least 2, at least 3, at least 4, or all 5 p53 epitopes and/or analogs from Table 6. The composition may comprise or consist of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, or all 14 epitopes and/or analogs from Table 16a; at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or all 20 epitopes and/or analogs from Table 16b; at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, or all 28 epitopes and/or analogs from Table 16c; at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or all 25 epitopes and/or analogs from Table 16d. The composition may comprise or consist of at least 1, at least 2, at least 3, at least 4, or all 5 epitopes and/or analogs from Table 17a; at least 1, at least 2, at least 3, at least 4, at least 5, or all 6 epitopes and/or analogs from Table 17b; at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, or all 14 epitopes and/or analogs from Table 17c; at least 1, at least 2, at least 3, or all 4 epitopes and/or analogs from Table 17d. The composition may comprise or consist of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, or all 27 epitopes and/or analogs from Table 18a; at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, or all 24 epitopes and/or analogs from Table 18b; at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, or all 28 epitopes and/or analogs from Table 18c; at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or all 10 epitopes and/or analogs from Table 18d. The composition may comprise or consist of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, at least 43, at least 44, or all 45 epitopes and/or analogs from Table 19a; at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or all 21 epitopes and/or analogs from Table 19b; at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, or all 40 epitopes and/or analogs from Table 19c; at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, or all 9 epitopes and/or analogs from Table 19d. The composition may comprise or consist of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, or at least 26 of the epitopes and/or analogs from Table 26, 27, 28, 29, or 30.

The composition may preferably comprise or consist of at least 1 or all 2 CEA epitopes/analogs of Table 9; at least 1 or all 2 HER2/neu epitopes/analogs of Table 9; at least 1 or all 2 MAGE2/3 epitopes/analogs of Table 9; at least 1 or all 2 p53 epitopes/analogs of Table 9. The composition may preferably comprise or consist of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, or all 7 CEA epitopes/analogs of Table 20; at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, or all 9 HER2/neu epitopes/analogs of Table 20; at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, or all 8 MAGE2/3 epitopes/analogs of Table 20; at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, or all 7 p53 epitopes/analogs of Table 20. The composition may preferably comprise or consist of at least the CEA epitope/analog of Table 21; at least the HER2/neu epitope/analog of Table 21; at least 1, at least 2, at least 3, or all 4 MAGE2/3 epitopes/analogs of Table 21; at least the p53 epitope/analog of Table 21. The composition may preferably comprise or consist of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, or all 8 CEA epitopes/analogs of Table 22; at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, or all 9 HER2/neu epitopes/analogs of Table 22; at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, or all 8 MAGE2/3 epitopes/analogs of Table 22; at least 1, at least 2, at least 3, at least 4, or all 5 p53 epitopes/analogs of Table 22. The composition may preferably comprise or consist of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 9, qat least 10, at least 11, or all 12 CEA epitopes/analogs of Table 23; at least 1, at least 2, at least 3, at least 4, at least 5, or all 6 HER2/neu epitopes/analogs of Table 23; at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, or all 13 MAGE2/3 epitopes/analogs of Table 23; at least 1, or all 2 p53 epitopes/analogs of Table 23.

The composition may comprise or consist of the combinations above and below, and may also exclude any one or several epitopes and/or analogs selected from those in Tables 6, 9, 10 (SEQ ID Nos:1-25), 16a-23 (SEQ ID NOS:42-362) and 26-30 (SEQ ID Nos:368-745). Epitopes/analogs which may preferably be excluded from composition of the invention are SEQ ID Nos:42, 60, 62, 67, 82, 86, 101, 116, 153, 362, 230, 265, 290, 321, 334, and 345.

The composition of the invention may comprise or consist of combinations of epitopes and/or analogs including:

A3 CEA combinations such as: (a) SEQ ID NOs:42, 44, 46, 51, 52, 54, and 55; (b) SEQ ID NOs: 44, 46, 51, 52, 54, and 55; (c) SEQ ID NOs:46, 51, 52, 54, and 55; (d) SEQ ID NOs: 51, 52, 54, and 55; (e) SEQ ID NO: 52, 54, and 55; (f) SEQ ID NO: 54 and 55;

(g) SEQ ID NO: 44, 46, 51, 52, and 54; (h) SEQ ID NO: 44, 46, 51, and 52; (i) SEQ ID NO: 44, 46, and 51; and 0) SEQ ID NO: 44 and 46;

(l) SEQ ID NO: 44, 51, 52, 54, and 55; (m) SEQ ID NO: 44, 46, 52, 54, and 55; (n) SEQ ID NO: 44, 46, 51, 54, and 55; and (O) SEQ ID NO: 44, 46, 51, 52, and 55;

A3 HER2/neu combinations such as: (a) SEQ ID NO:57, 60, 62, 67, 68, 69, 70, 73, and 75; (b) SEQ ID NO: 60, 62, 67, 68, 69, 70, 73, and 75; (c) SEQ ID NO: 62, 67, 68, 69, 70, 73, and 75; (d) 67, 68, 69, 70, 73, and 75; (e) 68, 69, 70, 73, and 75; (f) SEQ ID NO: 69, 70, 73, and 75;

(g) SEQ ID NO: 70, 73, and 75; (h) SEQ ID NO: 73 and 75;

(i) SEQ ID NO: 57, 60, 62, 67, 68, 69, 70, and 73; (j) SEQ ID NO: 57, 60, 62, 67, 68, 69, and 70; (k) SEQ ID NO: 57, 60, 62, 67, 68, and 69; (l) SEQ ID NO: 57, 60, 62, 67, and 68;

(m) SEQ ID NO: 57, 60, 62, and 67; (n) SEQ ID NO: 57, 60, and 62; (O) SEQ ID NO: 57 and 60; (p) SEQ ID NO: 57, 68, 69, 70, 73, and 75; (q) SEQ ED NO: 57, 60, 68, 69, 70, 73, and 75; (r) SEQ ID NO: 57, 60, 62, 69, 70, 73, and 75, and (s) SEQ ID NO: 57, 60, 62, 67, 68, 73, and 75;

A3 MAGE2/3 combinations such as: (a) SEQ ID NO:82, 90, 91, 96, 99, 102, and 103; (b) SEQ ID NO: 90, 91, 96, 99, 102, and 103; (c) SEQ ID NO: 91, 96, 99, 102, and 103; (d) SEQ ID NO: 96, 99, 102, and 103; (e) SEQ ID NO: 99, 102, and 103;

(f) SEQ ID NO: 102 and 103;

(g) SEQ ID NO: 77, 82, 90, 91, 96, 99, and 102; (h) SEQ ED NO: 77, 82, 90, 91, 96, and 99; (i) SEQ ID NO: 77, 82, 90, 91, and 96; (j) SEQ ID NO: 77, 82, 90, and 91; (k) SEQ ID NO: 77, 82, 90, and 91, 96, 99, 102, and 103; (l) SEQ ID ON: 77, 82, and 90; and (m) SEQ ID NO: 77 and 82;

A3 p53 combinations such as: (a) SEQ ID NO: 107, 111, 114, 116, 119, and 124; (b) SEQ ID NO: 111, 114, 116, 119, and 124; (c) SEQ ID NO: 114, 116, 119, and 124; (d) SEQ ID NO: 116, 119, and 124; (e) SEQ ID NO: 119 and 124;

(f) SEQ ID NO:104, 107, 111, 114, 116, and 119; (g) SEQ ID NO:104, 107, 111, 114, and 116; (h) SEQ ID NO:104, 107, 111, and 114; (i) SEQ ID NO:104, 107, and 111; (j) SEQ ID NO:104 and 107;

(k) SEQ ID NO:104, 111, 114, 116, 119, and 124; (l) SEQ ID NO:104, 107, 114, 116, 119, and 124; (m) SEQ ID NO:104, 107, 111, 116, 119, and 124; (n) SEQ ID NO:104, 107, 111, 114, 116, and 124;

B7 MAGE2/3 combinations such as: (a) SEQ ID NO: 146, 153, and 364; (b) SEQ ID NO: 153 and 364; (d) SEQ ID NO: 140, 146, and 153; (e) SEQ ID NO: 140, 146, and 364;

B7 combinations such as: (a) SEQ ID NO:133, 136, 140, 146, 153, and 155; (B) SEQ ID NO: 136, 140, 146, 153, and 155; (c) SEQ ID NO: 140, 146, 153, and 155; (d) —SEQ ID NO: 153 and 155;

A1 CEA combinations such as: (a) SEQ ID NO: 167, 170, 172, 178, 180, 181, and 182; (b) SEQ ID NO: 170, 172, 178, 180, 181, and 182; (c) SEQ ID NO: 172, 178, 180, 181, and 182; (d) SEQ ID NO: 178, 180, 181, and 182; (e) SEQ ID NO:180, 181, and 182; (f) SEQ ID NO: 181 and 182; (g) SEQ ID NO: 161, 167, 170, 172, 178, 180, and 181; (h) SEQ ID NO: 161, 167, 170, 172, 178, and 180; (i) SEQ ID NO: 161, 167, 170, 172, and 178; (j) SEQ ID NO: 161, 167, and 170; (k) SEQ ID NO: 181 and 182;

A1 HER2/neu combinations such as: (a) SEQ ID NO:188, 189, 191, 194, 198, 200, 201, and 208; (b) SEQ ID NO: 189, 191, 194, 198, 200, 201, and 208; (c) SEQ ID NO: 191, 194, 198, 200, 201, and 208; (d) SEQ ID NO: 194, 198, 200, 201, and 208; (e) SEQ ID NO: 198, 200, 201, and 208; (f) SEQ ID NO: 200, 201, and 208; (g) SEQ ID NO:201 and 208;

(h) SEQ ID NO:186, 188, 189, 191, 194, 198, 200, and 201; (i) SEQ ID NO:186, 188, 189, 191, 194, 198, and 200; 0) SEQ ID NO:186, 188, 189, 191, 194, and 198; (k) SEQ ID NO:186, 188, 189, 191, and 194; (l) SEQ ID NO:186, 188, 189, and 191; (m) SEQ ID NO:186, 188, and 189; (n) SEQ ID NO:186 and 188;

A1 MAGE2/3 combinations such as: (a) SEQ ED NO: 216, 219, 221, 228, 230, 234, and 236; (b) SEQ ID NO: 219, 221, 228, 230, 234, and 236; (c) SEQ ID NO: 221, 228, 230, 234, and 236; (d) SEQ ID NO: 228, 230, 234, and 236; (e) SEQ ID NO: 230, 234, and 236; (f) SEQ ID NO: 234 and 236; (g) SEQ ID NO:211, 216, 219, 221, 228, 230, and 234; h) SEQ ID NO:211, 216, 219, 221, 228, and 230; (i) SEQ ID NO:211, 216, 219, 221, and 228; 0) SEQ ID NO:211, 216, 219, and 221; Qc) SEQ ID NO:211, 216, and 219; (l) SEQ ID NO:211 and 216; (m) SEQ ID NO:211, 216, 219, 221, 228, 234, and 236;

A1 p53 combinations such as: (a) SEQ ID NO: 239, 240, 242, and 246; (b) SEQ ID NO: 240, 242, and 246; (c) SEQ ID NO: 242 and 246; (d) SEQ ID NO:238, 239, 240, and 242; (e) SEQ ID NO:238, 239, and 240; (f) SEQ ID NO:238 and 239; (g) SEQ ID NO:238, 240, 242, and 246; (h) SEQ ID NO:238, 239, 242, and 246; (i) SEQ ID NO:238, 239, 240, and 246;

A24 CEA combinations such as: (a) SEQ ID NO: 263, 265, 269, 272, 278, 279, 281, 282, 285, 287, and 290; (b) SEQ ID NO: 265, 269, 272, 278, 279, 281, 282, 285, 287, and 290; (c) SEQ ID NO: 269, 272, 278, 279, 281, 282, 285, 287, and 290; (d) SEQ ID NO: 272, 278, 279, 281, 282, 285, 287, and 290; (e) SEQ ID NO: 278, 279, 281, 282, 285, 287, and 290; (f) SEQ ID NO: 279, 281, 282, 285, 287, and 290; (g) SEQ ID NO: 281, 282, 285, 287, and 290; (h) SEQ ID NO: 282, 285, 287, and 290; (i) SEQ ID NO: 285, 287, and 290; (j) SEQ ID NO: 287 and 290; (k) SEQ ID NO:256, 263, 265, 269, 272, 278, 279, 281, 282, 285, and 287; (l) SEQ ID NO:256, 263, 265, 269, 272, 278, 279, 281, 282, and 285; (m) SEQ ID NO:256, 263, 265, 269, 272, 278, 279, 281, and 282; (n) SEQ ID NO:256, 263, 265, 269, 272, 278, 279, and 281; (o) SEQ ID NO:256, 263, 265, 269, 272, 278, and 279; (p) SEQ ID NO:256, 263, 265, 269, 272, and 278; (q) SEQ ID NO:256, 263, 265, 269, and 272; (r) SEQ ID NO:256, 263, 265, and 269; (s) SEQ ID NO:256, 263, and 265; (t) SEQ ID NO:256 and 263; (u) SEQ ID NO:256, 263, 269, 272, 278, 279, 281, 282, 285, and 287;

A24 HER2/neu combinations such as: (a) SEQ ID NO: 293, 304, 305, 308, and 310; (b) SEQ ID NO: 304, 305, 308, and 310; (c) SEQ ID NO: 305, 308, and 310; (d) SEQ ID NO: 308 and 310; (e) SEQ ID NO:292, 293, 304, 305, and 308; (f) SEQ ID NO:292, 293, 304, and 305; (g) SEQ ID NO:292, 293, and 304; (h) SEQ ID NO:292 and 293; (i) SEQ ID NO:292, 304, 305, 308, and 310; (j) SEQ ID NO:292, 293, 305, 308, and 310; (k) SEQ ID NO:292, 293, 304, 308, and 310; (l) SEQ ID NO:292, 293, 304, 305, and 310;

A24 MAGE2/3 combinations such as: (a) SEQ ID NO: 321, 324, 325, 331, 332, 333, 334, 335, 336, 344, 345, and 351; (b) SEQ ID NO: 324, 325, 331, 332, 333, 334, 335, 336, 344, 345, and 351; (c) SEQ ID NO: 325, 331, 332, 333, 334, 335, 336, 344, 345, and 351; (d) SEQ ID NO: 331, 332, 333, 334, 335, 336, 344, 345, and 351; (e) SEQ ID NO: 332, 333, 334, 335, 336, 344, 345, and 351; (f) SEQ ID NO: 333, 334, 335, 336, 344, 345, and 351; (g) SEQ ID NO: 333, 334, 335, 336, 344, 345, and 351; (h) SEQ ID NO: 334, 335, 336, 344, 345, and 351; (i) SEQ ID NO: 335, 336, 344, 345, and 351; (j) SEQ ID NO: 336, 344, 345, and 351; (k) SEQ ID NO: 344, 345, and 351; (l) SEQ ID NO:345 and 351;

(m) SEQ ID NO:316, 321, 324, 325, 331, 332, 333, 334, 335, 336, 344, and 345; (n) SEQ ID NO:316, 321, 324, 325, 331, 332, 333, 334, 335, 336, and 344; (o) SEQ ID NO:316, 321, 324, 325, 331, 332, 333, 334, 335, and 336; (p) SEQ ID NO:316, 321, 324, 325, 331, 332, 333, 334, and 335; (q) SEQ ID NO:316, 321, 324, 325, 331, 332, 333, and 334; (r) SEQ ID NO:316, 321, 324, 325, 331, 332, and 333; (s) SEQ ID NO:316, 321, 324, 325, 331, and 332; (t) SEQ ID NO:316, 321, 324, 325, and 331; (u) SEQ ID NO:316, 321, 324, and 325; (v) SEQ ID NO:316, 321, and 324; (w) SEQ ID NO:316 and 321; (x) SEQ ID NO:316, 324, 325, 331, 332, 333, 335, 336, 344, and 351;

A24 p53 combinations such as: SEQ ID NO:356 and 361;

B44 CEA combinations such as: (a) SEQ ID NO:368, 369, 390, 399, and 403; (b) SEQ ID NO:369, 370, 375, 376, 377, and 420; and (c) SEQ ID NO:370, 375, 379, 386, and 429;

B44 HER2/neu combinations such as: (a) SEQ ID NO:432, 435, 436, 443, 448, 460, 466, 467, and 488; (b) SEQ ID NO: 439, 473, 490, and 499; (c) SEQ ID NO:432, 433, 440, 441, 447, 456, 459, and 471; (d) SEQ ID NO: 477, 490, 499, 508, 527, and 535;

B44 MAGE2 combinations such as: (a) SEQ ID NO: 645, 646, 647, 653, 665, 670, 698, 718, and 716; (b) SEQ ID NO: 663, 688, 692, and 701; (c) SEQ ID NO:648, 655, 669, 677, 691, and 700; (d) SEQ ID NO: 651 and 673;

B44 MAGE3 combinations such as: (a) SEQ ID NO: 719, 720, 726, 732, and 740; (b) SEQ ID NO: 721, 725, 726, and 737; (c) SEQ ED NO: 726, 739, and 744; (d) SEQ ID NO: 722, 723, 728 and 735; (e) SEQ ID NO: 720, 728, 731, 736, and 741;

B44 p53 combinations such as: (a) SEQ ID NO: 598, 602, 603, and 617; (b) SEQ ID NO: 589, 599, 600, and 605; (c) SEQ ID NO:600, 603, 604, and 607; (d) SEQ ID NO: 601, 602, 604, and 609;

A2 combinations such as: (a) SEQ ID NO: 6, 8, 16, 18, 22, 23, and 24; (b) SEQ ID NO: 8, 16, 18, 22, 23, and 24; (c) SEQ ID NO: 16, 18, 22, 23, and 24; (d) SEQ ID NO: 18, 22, 23, and 24; (e) SEQ ID NO: 23 and 24; (f) SEQ ID NO: 1, 19, 3, and 4; (g) SEQ ID NO: 2, 6, 8, 16, 18, 22, and 23; (h) SEQ ID NO: 2, 6, 8, 16, 18, and 22; (i) SEQ ID NO: 2, 6, 8, 16, 18, and 22; (j) SEQ ID NO: 2, 6, 8, 16, and 18; (k) SEQ ID NO: 2, 6, 8, and 16; (l) SEQ ID NO: 2, 6, and 8; and (m) SEQ ID NO: 2 and 6; (n) SEQ ID NO:3, 4, 5, and 17; (o) SEQ ID NO: 20, 21, and 25; (p) SEQ ID NO: 1, 10, 17, and 25; (q) SEQ ID NO: 4, 5, 10, 17, and 25;

TAA combinations such as: (a) SEQ ID NO: 1, 17, 22, 104, 114, 133, 136, 146, 170, 189, 221, 310, 336, 361, and 399; (b) SEQ ID NO:111, 124, 133, 140, 155, 180, 194, 228, 246, and 281; (c) SEQ ID NO: 16, 18, 25, 43, 68, 117, 309, and 499; (d) SEQ ID NO: 48, 55, 97, 369, 409, and 512; (e) SEQ ID NO: 55, 99, 135, 238, and 602; (f) SEQ ID NO: 1, 58, 77, 104, 128, 166, 207, 240, 360, and 403; (g) SEQ ID NO:17, 50, 72, 130, 161, 199, 300, and 627; (h) SEQ ID NO: 10, 55, 82, 104, 198, 400, 433, and 501; (i) SEQ ID NO: 3, 22, 122, 196, 211, 301, 360, and 667; (j) SEQ ID NO: 1, 21, 44, 100, 207, 405, and 661.

Compositions of the invention may also comprise or consist of combinations of the above combinations, including:

A24 combinations such as: A24 CEA (a) and A24 HER2/neu (a); A24 CEA (a) and A24 MAGE2/3 (a); A24 CEA (a) and A24 p53; A24 CEA (c) and A24 HER2/neu (e); A24 CEA (i) and A24 MAGE2/3 (a); A24 CEA (n) and A24 p53 (k);

A3 combinations such as: A3 CEA (a) and A3 HER2/neu (a); A3 CEA (a) and A3 MAGE2/3 (a); A3 CEA (a) and A3 p53 (a); A3 CEA (d) and A3 HER2/neu (b); A3 CEA (f) and A3 MAGE2/3 (i); A3 CEA (e) and A3 p53 (a);

CEA combinations such as: A24 CEA (a) and A1 CEA (a); A24 CEA (b) and A1 CEA (a); A24 CEA (c) and A1 CEA (a); A24 CEA (c) and A1 CEA (a); A3 CEA (a) and A1 CEA (a); A3 CEA (b) and A1 CEA (a); A3 CEA (c) and A24 CEA (a); B7 CEA (c) and A1 CEA (a);

B7 CEA (a) and A3 CEA (a); B44 CEA (b) and A1 CEA (a); A3 CEA (e) and A1 CEA (g); A3 CEA (i) and A1 CEA (m);

A1 CEA (a), (b) (c), (d), (e), (f) (g), (h), (i), (j), or (k) epitopes/analogs, and A3 CEA (a), (b) (c), (d), (e), (t) (g), (h), (i), (j), or (k) epitopes/analogs, and B7 CEA (a), (b) (c), (d), (e), (f) (g), (h), (i), (j), or (k) epitopes/analogs, and A3 p53 (a), (b) (c), (d), (e), (f) (g), (h), (i), (j), or (k) epitopes/analogs, and B44 MAGE2 (a), (b) (c), (d), (e), (f) (g), (h), (i), (j), or (k) epitopes/analogs, and A3 MAGE2 (a), (b) (c), (d), (e), (f) (g), (h), (i), (j), or (k) epitopes/analogs; A24 CEA (a), (b) (c), (d), (e), (f) (g), (h), (i), (j), or (k) epitopes/analogs, and A2 CEA (a), (b) (c), (d), (e), (f) (g), (h), (i), (j), or (k) epitopes/analogs, and B7 MAGE3 (a), (b) (c), (d), (e), (f) (g), (h), (i), (j), or (k) epitopes/analogs, and B44 p53 (a), (b) (c), (d), (e), (f) (g), (h), (i), (j), or (k) epitopes/analogs; A3 CEA (a), (b) (c), (d), (e), (f) (g), (h), (i), (j), or (k) epitopes/analogs, and B7 p53 (a), (b) (c), (d), (e), (f), (g), (h), (i), (j), or (k) epitopes/analogs, and B44 MAGE3 (a), (b) (c), (d), (e), (f) (g), (h), (i), (j), or (k) epitopes/analogs, and A24 HER2/neu (a), (b) (c), (d), (e), (f) (g), (h), (i), (j), or (k) epitopes/analogs.

Compositions of the invention may comprise polynucleotides encoding the above peptides, and/or combinations of polynucleotides encoding the above combinations of peptides.

The composition can comprise at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, at least 43, at least 44, at least 44, at least 45, at least 46, at least 47, at least 48, at least 49, at least 50, peptides or polynucleotides selected from those described above or below. At least one of the one or more peptides can be a heteropolymer or a homopolymer. Additionally, the composition can comprise a CTL and/or HTL epitope, which can be derived from a tumor-associated antigen. The additional epitope can also be a PanDR binding molecule, (e.g., a PADRE® universal helper T cell epitope).

Optional components include excipients, diluents, proteins such as peptides comprising a CTL epitope, and/or an HTL epitope such as a pan-DR binding peptide (e.g., a PADRE® universal helper T cell epitope), and/or a carrier, polynucleotides encoding such proteins, lipids, or liposomes, as well as other components described herein. There are numerous embodiments of compositions in accordance with the invention, such as a cocktail of one or more peptides and/or polynucleotides; one or more peptides and/or analogs and one or more CTL and/or HTL epitopes; and/or nucleic acids that encode such peptides, e.g., minigenes.

Compositions may comprise one or more peptides (or polynucleotides such as minigenes) of the invention, along with one or more other components as described above and herein. “One or more” refers to any whole unit integer from 1-150, e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, or 150 peptides, polynucleotides, or other components.

Compositions of the invention may be, for example, polynucleotides or polypeptides of the invention combined with or complexed to cationic lipid formulations; lipopeptides (e.g., Vitiello, A. et al., J. Clin. Invest. 95:341, 1995), encapsulated e.g., in poly(DL-lactide-co-glycolide) (“PLG”) microspheres (see, e.g., Eldridge, et al., Molec. Immunol. 28:287-294, 1991: Alonso et al., Vaccine 12:299-306, 1994; Jones et al., Vaccine 13:675-681, 1995); peptide compositions contained in immune stimulating complexes (ISCOMS) (see, e.g., Takahashi et al., Nature 344:873-875, 1990; Hu et al., Clin Exp Immunol. 113:235-243, 1998); multiple antigen peptide systems (MAPs) (see e.g., Tam, J. P., Proc. Natl. Acad. Sci. U.S.A. 85:5409-5413, 1988; Tam, J. P., J. Immunol. Methods 196:17-32, 1996); viral, bacterial, or, fungal delivery vectors (Perkus, M. E. et al., In: Concepts in vaccine development, Kaufmann, S. H. E., ed., p. 379, 1996; Chakrabarti, S. et al., Nature 320:535, 1986; Hu, S. L. et al., Nature 320:537, 1986; Kieny, M.-P. et al., AIDS Bio/Technology 4:790, 1986; Top, F. H. et al., J. Infect. Dis. 124:148, 1971; Chanda, P. K. et al., Virology 175:535, 1990); particles of viral or synthetic origin (e.g., Kofler, N. et al., J. Immunol. Methods. 192:25, 1996; Eldridge, J. H. et al., Sem. Hematol. 30:16, 1993; Falo, L. D., Jr. et al., Nature Med. 7:649, 1995); adjuvants (e.g., incomplete Freund's adjuvant) (Warren, H. S., Vogel, F. R., and Chedid, L. A. Annu. Rev. Immunol. 4:369, 1986; Gupta, R. K. et al., Vaccine 11:293, 1993); liposomes (Reddy, R. et al., J. Immunol. 148:1585, 1992; Rock, K. L., Immunol. Today 17:131, 1996); or, particle-absorbed cDNA or other polynucleotides of the invention (Ulmer, J. B. et al., Science 259:1745, 1993; Robinson, H. L., Hunt, L. A., and Webster, R. G., Vaccine 11:957, 1993; Shiver, J. W. et al., In: Concepts in vaccine development, Kaufmann, S. H. E., ed., p. 423, 1996; Cease, K. B., and Berzofsky, J. A., Annu. Rev. Immunol. 12:923, 1994 and Eldridge, J. H. et al., Sem. Hematol. 30:16, 1993), etc. Toxin-targeted delivery technologies, also known as receptor mediated targeting, such as those of Avant Immunotherapeutics, Inc. (Needham, Mass.) or attached to a stress protein, e.g., HSP 96 (Stressgen Biotechnologies Corp., Victoria, BC, Canada) can also be used.

Compositions of the invention comprise polynucleotide-mediated modalities. DNA or RNA encoding one or more of the peptides of the invention can be administered to a patient. This approach is described, for instance, in Wolff et. al., Science 247:1465 (1990) as well as U.S. Pat. Nos. 5,580,859; 5,589,466; 5,804,566; 5,739,118; 5,736,524; 5,679,647; and, WO 98/04720. Examples of DNA-based delivery technologies include “naked DNA”, facilitated (bupivicaine, polymers (e.g., PVP, PINC, etc.), peptide-mediated) delivery, cationic lipid complexes, and particle-mediated (“gene gun”) or pressure-mediated delivery (see, e.g., U.S. Pat. No. 5,922,687). Accordingly, peptides of the invention can be expressed by viral or bacterial vectors. Examples of expression vectors include attenuated viral hosts, such as Modified Vaccinia Ankara (MVA) (e.g., Bavarian Noridic), vaccinia or fowlpox. For example, vaccinia virus is used as a vector to express nucleotide sequences that encode the peptides of the invention. Upon introduction into an acutely or chronically infected host or into a non-infected host, the recombinant vaccinia virus expresses the immunogenic peptide, and thereby elicits an immune response. Vaccinia vectors and methods useful in immunization protocols are described in, e.g., U.S. Pat. No. 4,722,848. Another vector is BCG (Bacille Calmette Guerin). BCG vectors are described in Stover et al., Nature 351:456-460 (1991). A wide variety of other vectors useful for therapeutic administration or immunization of the peptides of the invention, e.g. adeno and adeno-associated virus vectors, alpha virus vectors, retroviral vectors, Salmonella typhi vectors, detoxified anthrax toxin vectors, and the like, are apparent to those skilled in the art from the description herein.

In certain embodiments, components that induce T cell responses are combined with components that induce antibody responses to the target antigen of interest. A preferred embodiment of such a composition comprises class I and class II epitopes in accordance with the invention. Alternatively, a composition comprises a class I and/or class II epitope in accordance with the invention, along with a PADRE® molecule Epimmune, San Diego, Calif.).

Compositions of the invention can comprise antigen presenting cells, such as dendritic cells. Antigen presenting cells, e.g., dendritic cells, may be transfected, e.g., with a polynucleotide such as a minigene construct in accordance with the invention, in order to elicit immune responses. The peptide can be bound to an HLA molecule on the antigen-resenting cell, whereby when an HLA-restricted cytotoxic T lymphocyte (CTL) is present, a receptor of the CTL binds to a complex of the HLA molecule and the peptide.

The compositions of the invention may also comprise antiviral drugs such as interferon-α, or immune adjuvants such as IL-12, GM-CSF, etc.

Compositions may comprise an HLA heavy chain, β₂-microglobulin, streptavidin, and/or biotin. The streptavidin may be fluorescently labeled. Compositions may comprise tetramers (see e.g., U.S. Pat. No. 5,635,363; Science 274:9496 (1996)). A tetramer composition comprising an HLA heavy chain, β₂-Microglobulin, streptavidin, and biotin. The streptavidin may be fluorescently labeled. Compositions may also comprise dimers. A dimer composition comprises as MHC molecule and an Ig molecule (see e.g., PNAS 95:7568-73 (1998)).

In some embodiments it may be desirable to include in the compositions of the invention at least one component which primes cytotoxic T lymphocytes. Lipids have been identified as agents capable of priming CTL in vivo against viral antigens. For example, palmitic acid residues can be attached to the ε- and α-amino groups of a lysine residue and then linked, e.g., via one or more linking residues such as Gly, Gly-Gly-, Ser, Ser-Ser, or the like, to an immunogenic peptide. The lipidated peptide can then be administered either directly in a micelle or particle, incorporated into at liposome, or emulsified in an adjuvant, e.g., incomplete Freund's adjuvant. A preferred composition comprises palmitic acid attached to ε- and α-amino groups of Lys, which is attached via linkage, e.g., Ser-Ser, to the amino terminus of the peptide.

As another example of lipid priming of CTL responses, E. coli lipoproteins, such as tripalmitoyl-S-glycerylcysteinlyseryl-serine (P₃CSS) can be used to prime virus specific CTL when covalently attached to an appropriate peptide (see, e.g., Deres, et al., Nature 342:561, 1989). Peptides of the invention can be coupled to P₃CSS, for example, and the lipopeptide administered to an individual to specifically prime a CTL response to the target antigen. Moreover, because the induction of neutralizing antibodies can also be primed with P₃CSS-conjugated epitopes, two such compositions can be combined to more effectively elicit both humoral and cell-mediated responses.

Another preferred embodiment is a composition comprising one or more peptides of the invention emulsified in IFA.

Compositions of the invention may also comprise CTL and/or HT peptides. Such CTL and HTL peptides can be modified by the addition of amino acids to the termini of a peptide to provide for ease of linking peptides one to another, for coupling to a carrier support or larger peptide, for modifying the physical or chemical properties of the peptide or oligopeptide, or the like. Amino acids such as tyrosine, cysteine, lysine, glutamic or aspartic acid, or naturally or unnaturally occuring amino acid residues, can be introduced at the carboxyl- or amino-terminus of the peptide or oligopeptide, particularly class I peptides. However, it is to be noted that modification at the carboxyl terminus of a CTL epitope may, in some cases, alter binding characteristics of the peptide. In addition, the peptide or oligopeptide sequences can differ from the natural sequence by being modified by terminal-NH₂ acylation, e.g., by alkanoyl (C₁-C₂₀) or thioglycolyl acetylation, terminal-carboxylamidation, e.g., ammonia, methylamine, etc. In some instances these modifications may provide sites for linking to a support or other molecule. CTL and HTL epitopes may comprise additional amino acids, such as those described above including spacers.

A further embodiment of a composition in accordance with the invention is an antigen presenting cell that comprises one or more peptides in accordance with the invention. The antigen presenting cell can be a “professional” antigen presenting cell, such as a dendritic cell. The antigen presenting cell can comprise the peptide of the invention by any means known or to be determined in the art. Such means include pulsing of dendritic cells with one or more individual peptides, by nucleic acid administration such as ballistic nucleic acid delivery or by other techniques in the art for administration of nucleic acids, including vector-based, e.g. viral vector, delivery of nucleic acids.

Compositions may comprise carriers. Carriers that can be used with compositions of the invention are well known in the art, and include, e.g., thyroglobulin, albumins such as human serum albumin, tetanus toxoid, polyamino acids such as poly L-lysine, poly L-glutamic acid, influenza virus proteins, hepatitis B virus core protein, and the like.

The compositions (e.g. pharmaceutical compositions) can contain a physiologically tolerable diluent such as water, or a saline solution, preferably phosphate buffered saline. Additionally, as disclosed herein, CTL responses can be primed by conjugating peptides of the invention to lipids, such as tripalmitoyl-S-glyceryl-cysteinyl-seryl-serine (P₃CSS).

Compositions of the invention may be pharmaceutically acceptable compositions. Pharmaceutical compositions preferably contain an immunologically effective amount of one or more peptides and/or polynucleotides of the invention, and optionally one or more other components which are pharmaceutically acceptable. A preferred composition comprises one or more peptides of the invention and IFA. A more preferred composition of the invention comprises one or more peptides of the invention, one or more peptides, and IFA.

Upon immunization with a peptide and/or polynucleotide and/or composition in accordance with the invention, via injection (e.g., SC, ID, M), aerosol, oral, transdermal, transmucosal, intrapleural, intrathecal, or other suitable routes, the immune system of the host responds to the vaccine by an immune response comprising the production of antibodies, CTLs and/or HTLs specific for the desired antigen(s). Consequently, the host becomes at least partially immune to subsequent exposure to the TAA(s), or at least partially, resistant to further development of TAA-bearing cells and thereby derives a prophylactic or therapeutic benefit.

Furthermore, the peptides, primers, and epitopes of the invention can be used in any desired immunization or administration regimen; e.g., as part of periodic vaccinations such as annual vaccinations as in the veterinary arts or as in periodic vaccinations as in the human medical arts, or as in a prime-boost regime wherein an inventive vector or recombinant is administered either before or after the administration of the same or of a different epitope of interest or recombinant or vector expressing such as a same or different epitope of interest (including an inventive recombinant or vector expressing such as a same or different epitope of interest), see, e.g., U.S. Pat. Nos. 5,997,878; 6,130,066; 6,180,398; 6,267,965; and 6,348,450. An useful viral vector of the present invention is Modified Vaccinia Ankara (MVA) (e.g., Bavarian Noridic (MVA-BN)).

Recent studies have indicated that a prime-boost protocol, whereby immunization with a poxvirus recombinant expressing a foreign gene product is followed by a boost using a purifired subunit preparation form of that gene product, elicits an enhanced immune response relative to the response elicited with either product alone. Human volunteers immunized with a vaccinia recombinant expressing the HIV-1 envelope glycoprotein and boosted with purified HIV-1 envelope glycoprotein subunit preparation exhibit higher HV-1 neutralizing antibody titers than individuals immunized with just the vaccinia recombinant or purified envelope glycoprotein alone (Graham et al., J. Infect. Dis., 167:533-537 (1993); Cooney et al, Proc. Natl. Acad. Sci. USA, 90:1882-1886 (1993)). Humans immunized with two injections of an ALVAC-HIV-1 env recombinant (vCP125) failed to develop HIV specific antibodies. Boosting with purified rgp160 from a vaccinia virus recombinant resulting in detectable HIV-1 neutralizing antibodies. Furthermore, specific lymphocyte T cell proliferation to rgp160 was clearly increased by the boost with rgp160. Envelope specific cytotoxic lymphocyte activity was also detected with this vaccination regimen (Pialoux et al., AIDS Res. and Hum. Retroviuses, 11:272-381 (1995)). Marcaques immunized with a vaccinia recombinant expressing the simian immunodeficiency virus (SIV) envelope glycoprotein and boosted with SIV envelope glycoprotein from a baculovirus recombinant are protected against SIV challenge (Hu et al., AID Res. and Hum. Retroviruses, 3:615-620 (1991); Hu et al., Science 255:456-459 (1992)). In the same fashion, purified HCMVgB protein can be used in prime-boost protocols with NYVAC or ALVAC-GB recombinants.

In certain embodiments, the polynucleotides are complexed in a liposome preparation. Liposomal preparations for use in the instant invention include cationic (positively charged), anionic (negatively charged) and neutral preparations. However, cationic liposomes are particularly preferred because a tight charge complex can be formed between the cationic liposome and the polyanionic nucleic acid. Cationic liposomes have been shown to mediate intracellular delivery of plasmid DNA (Felgner et al., Proc. Natl. Acad. Sci. USA 84:74137416 (1987), which is herein incorporated by reference); mRNA Malone et al., Proc. Natl. Acad. Sci. USA 86:60776081 (1989), which is herein incorporated by reference); and purified transcription factors (Debs et al., J. Biol. Chem. 265:1018910192 (1990), which is herein incorporated by reference), in functional form.

Cationic liposomes are readily available. For example, N-[12,3-dioleyloxy)-propyl]-N,N,N-triethylammonium (DOTMA) liposomes are particularly useful and are available under the trademark Lipofectin, from GIBCO BRL, Grand Island, N.Y. (See, also, Felgner et al., Proc. Natl. Acad. Sci. USA 84:74137416 (1987)). Other commercially available liposomes include transfectace (DDAB/DOPE) and DOTAP/DOPE (Boehringer).

Other cationic liposomes can be prepared from readily available materials using techniques well known in the art. See, e.g. PCT Publication No. WO 90/11092 for a description of the synthesis of DOTAP (1,2-bis(oleoyloxy)-3-(trimethylammonio)propane) liposomes. Preparation of DOTMA liposomes is explained in the literature, see, e.g., P. Felgner et al., Proc. Natl. Acad. Sci. USA 84:74137417. Similar methods can be used to prepare liposomes from other cationic lipid materials.

Similarly, anionic and neutral liposomes are readily available, such as from Avanti Polar Lipids (Birmingham, Ala.), or can be easily prepared using readily available materials. Such materials include phosphatidyl, choline, cholesterol, phosphatidyl ethanolamine, dioleoylphosphatidyl choline (DOPC), dioleoylphosphatidyl glycerol (DOPG), dioleoylphoshatidyl ethanolamine (DOPE), among others. These materials can also be mixed with the DOTMA and DOTAP starting materials in appropriate ratios. Methods for making liposomes using these materials are well known in the art.

For example, commercially available dioleoylphosphatidyl choline (DOPC), dioleoylphosphatidyl glycerol (DOPG), and dioleoylphosphatidyl ethanolamine (DOPE) can be used in various combinations to make conventional liposomes, with or without the addition of cholesterol. Thus, for example, DOPG/DOPC vesicles can be prepared by drying 50 mg each of DOPG and DOPC under a stream of nitrogen gas into a sonication vial. The sample is placed under a vacuum pump overnight and is hydrated the 64′ following day with deionized water. The sample is then sonicated for 2 hours in a capped vial, using a Heat Systems model 350 sonicator equipped with an inverted cup (bath type) probe at the maximum setting while the bath is circulated at 15EC. Alternatively, negatively charged vesicles can be prepared without sonication to produce multilamellar vesicles or by extrusion through nucleopore membranes to produce unilamellar vesicles of discrete size. Other methods are known and available to those of skill in the art.

The liposomes can comprise multilamellar vesicles (MLVs), small unilamellar vesicles (SUVs), or large unilamellar vesicles (LUVs), with SUVs being preferred. The various liposome nucleic acid complexes are prepared using methods well known in the art. See, e.g., Straubinger et al., Methods of Immunology 101:512527 (1983). For example, MLVs containing nucleic acid can be prepared by depositing a thin film of phospholipid on the walls of a glass tube and subsequently hydrating with a solution of the material to be encapsulated. SUVs are prepared by extended sonication of MLVs to produce a homogeneous population of unilamellar liposomes. The material to be entrapped is added to a suspension of preformed MLVs and then sonicated. When using liposomes containing cationic lipids, the dried lipid film is resuspended in an appropriate solution such as sterile water or an isotonic buffer solution such as 10 mM Tris/NaCl, sonicated, and then the preformed liposomes are mixed directly with the DNA. The liposome and DNA form a very stable complex due to binding of the positively charged liposomes to the cationic DNA. SUVs find use with small nucleic acid fragments. LUVs are prepared by a number of methods, well known in the art. Commonly used methods include Ca²⁺-EDTA chelation (Papahadjopoulos et al., Biochim. Biophys. Acta 394:483 (1975); Wilson et al., Cell 17:77 (1979)); ether injection (Deamer, D. and Bangham, A., Biochim. Biophys. Acta 443:629 (1976); Ostro et al., Biochem. Biophys. Res. Commun. 76:836 (1977); Fraley et al., Proc. Natl. Acad. Sci. USA 76:3348 (1979)); detergent dialysis (Enoch, H. and Strittmatter, P., Proc. Natl. Acad. Sci. USA 76:145 (1979)); and reversephase evaporation (REV) (Fraley et al., J. Biol. Chem. 255:10431 (1980); Szoka, F. and Papahadjopoulos, D., Proc. Natl. Acad. Sci. USA 75:145 (1978); SchaeferRidder et al., Science 215:166 (1982)).

Generally, the ratio of DNA to liposomes will be from about 10:1 to about 1:10. Preferably, the ration will be from about 5:1 to about 1:5. More preferably, the ration will be about 3:1 to about 1:3. Still more preferably, the ratio will be about 1:1.

U.S. Pat. No. 5,676,954 reports on the injection of genetic material, complexed with cationic liposomes carriers, into mice. U.S. Pat. Nos. 4,897,355, 4,946,787, 5,049,386, 5,459,127, 5,589,466, 5,693,622, 5,580,859, 5,703,055, and international publication no. WO 94/9469 provide cationic lipids for use in transfecting DNA into cells and mammals. U.S. Pat. Nos. 5,589,466, 5,693,622, 5,580,859, 5,703,055, and international publication no. WO 94/9469 provide methods for delivering DNA-cationic lipid complexes to mammals.

Binding Affinity of Epitopes and Analogs for HLA Molecules

As indicated herein, the large degree of HLA polymorphism is an important factor to be taken into account with the epitope-based approach to developing therapeutics and diagnostics. To address this factor, epitope selection encompassing identification of peptides capable of binding at high or intermediate affinity to multiple HLA molecules is preferably utilized, most preferably these epitopes bind at high or intermediate affinity to two or more allele-specific HLA molecules. However, in some embodiments, it is preferred that all epitopes in a given composition bind to the alleles of a single HLA supertype or a single HLA molecule.

Epitopes and analogs of the invention preferably include those that have an IC₅₀ or binding affinity value for a class I HLA molecule(s) of 500 nM or better (i.e., the value is ≦500 nM). In certain embodiments of the invention, peptides of interest have an IC₅₀ or binding affinity value for a class I HLA molecule(s) of 200 nM or better. In certain embodiments of the invention, peptides of interest, such as A1 and A24 peptides, have an IC₅₀ or binding affinity value for a class I HLA molecule(s) of 100 nM or better. If HTL epitopes are included, they preferably are HTL epitopes that have an IC₅₀ or binding affinity value for class II HLA molecules of 1000 nM or better, (i.e., the value is ≦1,000 nM). For example, peptide binding is assessed by testing the capacity of a candidate peptide to bind to a purified HLA molecule in vitro. Peptides exhibiting high or intermediate affinity are then considered for further analysis. Selected peptides are generally tested on other members of the supertype family. In preferred embodiments, peptides that exhibit cross-reactive binding are then used in cellular screening analyses or vaccines.

The relationship between binding affinity for HLA class I molecules and immunogenicity of discrete peptide epitopes on bound antigens was determined for the first time by inventors at Epimmune. As disclosed in greater detail herein, higher HLA binding affinity is correlated with greater immunogenicity.

Greater immunogenicity can be manifested in several different ways. Immunogenicity corresponds to whether an immune response is elicited at all, and to the vigor of any particular response, as well as to the extent of a population in which a response is elicited. For example, a peptide might elicit an immune response in a diverse array of the population, yet in no instance produce a vigorous response. In accordance with these principles, close to 90% of high binding peptides have been found to elicit a response and thus be “immunogenic,” as contrasted with about 50% of the peptides that bind with intermediate affinity. (See, e.g., Schaeffer et al. PNAS (1988)) High affinity-binding class I peptides generally have an affinity of less than or equal to 100 nM. Moreover, not only did peptides with higher binding affinity have an enhanced probability of generating an immune response, the generated response tended to be more vigorous than the response seen with weaker binding peptides. As a result, less peptide is required to elicit a similar biological effect if a high affinity binding peptide is used rather than a lower affinity one. Thus, in some preferred embodiments of the invention, high affinity binding epitopes are used.

The correlation between binding affinity and immunogenicity was analyzed by the present inventors by two different experimental approaches (see, e.g., Sette, et al., J. Immunol. 153:5586-5592 (1994)). In the first approach, the immunogenicity of potential epitopes ranging in HLA binding affinity over a 10,000-fold range was analyzed in HLA-A*0201 transgenic mice. In the second approach, the antigenicity of approximately 100 different hepatitis B virus (HBV)-derived potential epitopes, all carrying A*0201 binding motifs, was assessed by using PBL from acute hepatitis patients. Pursuant to these approaches, it was determined that an affinity threshold value of approximately 500 nM (preferably 50 nM or less) determines the capacity of a peptide epitope to elicit a CTL response. These data are true for class I binding affinity measurements for naturally processed peptides and for synthesized T cell epitopes. These data also indicate the important role of determinant selection in the shaping of T cell responses (see, e.g. Schaeffer et al. Proc. Natl. Acad. Sci. USA 86:46494653 (1989)).

An affinity threshold associated with immunogenicity in the context of HLA class II (i.e., HLA DR) molecules has also been delineated (see, e.g., Southwood et al. J. Immunology 160:3363-3373 (1998), and U.S. Pat. No. 6,413,527, issued Jul. 2, 2002). In order to define a biologically significant threshold of HLA class II binding affinity, a database of the binding affinities of 32 DR-restricted epitopes for their restricting element (i.e., the HLA molecule that binds the epitope) was compiled. In approximately half of the cases (15 of 32 epitopes), DR restriction was associated with high binding affinities, i.e. binding affinity values of 100 nM or less. In the other half of the cases (16 of 32), DR restriction was associated with intermediate affinity (binding affinity values in the 100-1000 nM range). In only one of 32 cases was DR restriction associated with an IC₅₀ of 1000 nM or greater. Thus, 1000 nM is defined as an affinity threshold associated with immunogenicity in the context of DR molecules.

The binding affinity of peptides for HLA molecules can be determined as described in Example 3, below.

Epitope Binding Motifs and Supermotifs

Through the study of single amino acid substituted antigen analogs and the sequencing of endogenously bound, naturally processed peptides, critical residues required for allele-specific binding to HLA molecules have been identified. The presence of these residues in a peptide correlates with both the probability of binding and with binding affinity for HLA molecules.

The identification of motifs and/or supermotifs that correlate with high and intermediate affinity binding is important when identifying immunogenic peptide epitopes for the inclusion in a vaccine. Kast et al. (J. Immunol. 152:3904-3912 (1994)) have shown that motif-bearing peptides account for 90% of the epitopes that bind to allele-specific HLA class I molecules. In the Kast study, all possible 9 amino acid long peptides, each overlapping by eight amino acids, which cover the entire sequence of the E6 and E7 proteins of human papillomavirus type 16 were generated, which produced 240 peptides. All 240 peptides were evaluated for binding to five allele-specific HLA molecules that are expressed at high frequency among different ethnic groups. This unbiased set of peptides allowed an evaluation of the predictive values of HLA class I motifs. From the set of 240 peptides, 22 peptides were identified that bound to an allele-specific HLA molecule with high or intermediate affinity. Of these 22 peptides, 20 (i.e. 91%) were motif-bearing. Thus, this study demonstrated the value of motifs for identification of peptide epitopes to be included in a vaccine.

Accordingly, the use of motif-based identification techniques identifies approximately 90% of all potential epitopes in a target protein sequence. Without the disclosed motif analysis, the ability to practically identify immunogenic peptide(s) for use in diagnostics or therapeutics is seriously impaired.

Peptides, pharmaceutical compositions and vaccines of the present invention may also comprise epitopes that bind to MHC class II DR molecules. A greater degree of heterogeneity in both size and binding frame position of the motif, relative to the N- and C-termini of the peptide, exists for class II peptide ligands. This increased heterogeneity of HLA class II peptide ligands is due to the structure of the binding groove of the HLA class II molecule which, unlike its class I counterpart, is less physically constricted at both ends. Crystallographic analysis of HLA class II DRB*0101-peptide complexes to identify the residues associated with major binding energy identified those residues complexed with complementary pockets on the DRBI*0101 molecules. An important anchor residue engages the deepest hydrophobic pocket (see, e.g., Madden, D. R. Ann. Rev. Immunol. 13:587 (1995)) and is referred to as position 1 (P1). P1 may represent the N-terminal residue of a class II binding peptide epitope, but more typically is flanked towards the N-terminus by one or more residues. Other studies have also pointed to an important role for the peptide residue in the sixth position towards the C-terminus, relative to P1, for binding to various DR molecules. See, e.g., U.S. Pat. No. 5,736,142, and co-pending applications entitled Alteration Of Immune Responses Using Pan DR Binding Peptides, U.S. Ser. No. 09/709,774, filed Nov. 8, 2000 and 09/707,738, filed Nov. 6, 2000.

Thus, a large fraction of HLA class I and class II molecules can be classified into a relatively few supertypes, each respective supertype characterized by largely overlapping peptide binding repertoires, and consensus structures of the main peptide binding pockets. Thus, peptides of the present invention are preferably identified by any one of several HLA-specific amino acid motifs (see, e.g., Tables 2-4), or if the presence of the motif corresponds to the ability to bind several allele-specific HLA antigens, a supermotif (see, e.g., Tables 14-16d).

The primary anchor residues of the HLA class I peptide epitope supermotifs and motifs are summarized in Tables 2 and 14. The HLA class I motifs set out in Tables 2, 2a and 14 are particularly relevant to the invention claimed here. Primary and secondary anchor positions for HLA Class I are summarized in Table 3. Allele-specific HLA molecules that are comprised by the various HLA class I supertypes are listed in Table 5. In some cases, patterns of amino acid residues are present in both a motif and a supermotif. The relationship of a particular motif and any related supermotif is indicated in the description of the individual motifs.

Thus, the peptide motifs and supermotifs described below, and summarized in Tables 2-4 and 14, provide guidance for the identification and use of peptide epitopes in accordance with the invention.

HLA-A1 Supermotif

The HLA-A1 supermotif is characterized by the presence in peptide ligands of a small (T or S) or hydrophobic (L, I, V, or M) primary anchor residue in position 2, and an aromatic (Y, F, or W) primary anchor residue at the C-terminal position of the epitope. The corresponding family of HLA molecules that bind to the A1 supermotif (i.e., the HLA-A1 supertype) is comprised of at least A*0101, A*2601, A*2602, A*2501, A*2902 and A*3201 (Sette, et al. Immunogenetics 50:201 (1999)). Other allele-specific HLA molecules predicted to be members of the A1 superfamily are shown in Table 5.

HLA-A1 Motif

The HLA-A1 motif is characterized by the presence in peptide ligands of T, S, or M as a primary anchor residue at position 2 and the presence of Y as a primary anchor residue at the C-terminal position of the epitope. An alternative allele-specific A1 motif is characterized by a primary anchor residue at position 3 rather than position 2. This motif is characterized by the presence of D, E, A, or S as a primary anchor residue in position 3, and a Y as a primary anchor residue at the C-terminal position of the epitope (see, e.g., DiBrino et al., J. Immunol., 152:620, 1994; Kondo et al., Immunogenetics 45:249, 1997; and Kubo et al., J. Immunol. 152:3913, 1994 for reviews of relevant data).

HLA-A3 Supermotif

The HLA-A3 supermotif is characterized by the presence in peptide ligands of A, L, I, V, M, S, or, T as a primary anchor at position 2, and a positively charged residue, R or K, at the C-terminal position of the epitope, e.g. in position 9 of 9-mers (see, e.g., Sidney et al., Hum. Immunol. 45:79, 1996). Exemplary members of the corresponding family of HLA molecules (the HLA-A3 supertype) that bind the A3 supermotif include at least A*0301, A*1101, A*3101, A*3301, and A*6801. Other allele-specific HLA molecules predicted to be members of the A3 supertype are shown in Table 5.

HLA-A3 Motif

The HLA-A3 motif is characterized by the presence in peptide ligands of L, M, V, L S, A, T, F, C, G, or D as a primary anchor residue at position 2, and the presence of K, Y, R, H, F, or A as a primary anchor residue at the C-terminal position of the epitope (see, e.g., DiBrino et al., Proc. Natl. Acad. Sci USA 90:1508, 1993; and Kubo et al., J. Immunol. 152:3913-3924, 1994).

HLA-A24 Supermotif

The HLA-A24 supermotif is characterized by the presence in peptide ligands of an aromatic (F, W, or Y) or hydrophobic aliphatic (L, L V, M, or T) residue as a primary anchor in position 2, and Y, F, W, L, I, or M as primary anchor at the C-terminal position of the epitope (see, e.g., Sette and Sidney, Immunogenetics, 50:201-212, 1999). The corresponding family of HLA molecules that bind to the A24 supermotif (i.e., the A24 supertype) includes at least A*2402, A*3001, A*2301 and A*3002. Other allele-specific HLA molecules predicted to be members of the A24 supertype are shown in Table 5.

HLA-B7 Supermotif

The HLA-B7 supermotif is characterized by peptides bearing proline in position 2 as a primary anchor, and a hydrophobic or aliphatic amino acid (L, I, V, M, A, F, W, or Y) as the primary anchor at the C-terminal position of the epitope. The corresponding family of HLA molecules that bind the B7 supermotif (i.e., the HLA-B7 supertype) is comprised of at least twenty six HLA-B proteins including: B*0702, B*0703, B*0704, B*0705, B*1508, B*3501, B*3502, B*3503, B*3504, B*3505, B*3506, B*3507, B*3508, B*5101, B*5102, B*5103, B*5104, B*5105, B*5301, B*5401, B*5501, B*5502, B*5601, B*5602, B*6701, and B*7801 (see, e.g., Sidney, et al., J. Immunol. 154:247, 1995; Barber, et al., Curr. Biol. 5:179, 1995; Hill, et al., Nature. 360:434, 1992; Rammensee, et al., Immunogenetics 41:178, 1995 for reviews of relevant data). Other allele-specific HLA molecules predicted to be members of the B7 supertype are shown in Table 5.

HLA-B44 Supermotif

The HLA-B44 supermotif is characterized by the presence in peptide ligands of negatively charged (D or E) residues as a primary anchor in position 2, and a hydrophobic residues (F, W, Y, L M, T, L, A, or V) as the primary anchor of the C-terminal position of the epitope (see, e.g., Sidney et al. Immunol. Today 1 7:261, 1996). Exemplary members of the corresponding family of HLA molecules that bind to the B44 supermotif (i.e., the HLA-B44 supertype) includes at least: B*1801, B*4001(B60), B*4002 (B61), B*4402, B*4403, and B*4501. Other allele-specific HLA molecules predicted to be members of the B44 supertype are shown in Table 5.

HLA-A2 Supermotif

Primary anchor specificities for allele-specific HLA-A2.1 molecules (see, e.g., Falk et al., Nature 351:290-296 (1991); Hunt et al., Science 255:1261-1263 (1992); Parker et al., J. Immunol. 149:3580-3587 (1992); Ruppert et al., Cell 74:929-937 (1993)) and cross-reactive binding among HLA-A2 and -A28 molecules have been described. (See, e.g., Fruci et al., Human Immunol. 38:187-192 (1993); Tanigaki et al., Human Immunol. 39:155-162 (1994); del Guercio et al, J. Immunol. 154:685-693 (1995); Kast et al., J. Immunol. 152:39043912 (1994) for reviews of relevant data.) These primary anchor residues define the HLA-A2 supermotif; which when present in peptide ligands corresponds to the ability to bind several different HLA-A2 and -A28 molecules. The HLA-A2 supermotif comprises peptide ligands with L, L V, M, A, T, or Q as a primary anchor residue at position 2 and L, I, V, M, A, or T as a primary anchor residue at the C-terminal position of the epitope.

The corresponding family of HLA molecules (i.e., the HLA-A2 supertype that binds these peptides) is comprised of at least: A*0201, A*0202, A*0203, A*0204, A*0205, A*0206, A*0207, A*0209, A*0214, A*6802, and A*6901. Other allele-specific HLA molecules predicted to be members of the A2 superfamily are shown in Table 5. As explained in detail below, binding to each of the individual allele-specific HLA molecules can be modulated by substitutions at the primary anchor and/or secondary anchor positions, preferably choosing respective residues specified for the supermotif.

HLA-A*0201 Motif

An HLA-A2*0201 motif was determined to be characterized by the presence in peptide ligands of L or M as a primary anchor residue in position 2, and L or V as a primary anchor residue at the C-terminal position of a 9-residue peptide (see, e.g., Falk et al., Nature 351:290-296 (1991)) and was further found to comprise an I at position 2 and I or A at the C-terminal position of a nine amino acid peptide (see, e.g., Hunt et al., Science 255:1261-1263, Mar. 6, 1992; Parker et al., J. Immunol. 149:3580-3587 (1992)) and position 10 of a decamer peptide. The A*0201 allele-specific motif has also been defined by the present inventors to additionally comprise V, A, T, or Q as a primary anchor residue at position 2, and M or T as a primary anchor residue at the C-terminal position of the epitope (see, e.g., Kast et al., J. Immunol. 152:3904-3912, 1994).

Thus, the HLA-A*0201 motif comprises peptide ligands with L, I, V, M, A, T, or Q as primary anchor residues at position 2 and L, I, V, M, A, or T as a primary anchor residue at the C-terminal position of the epitope. For this motif-supermotif relationship the preferred and less preferred/tolerated residues that characterize the primary anchor positions of the HLA-A*0201 motif are identical to the residues describing the A2 supermotif. (For reviews of relevant data, see, e.g., del Guercio et al., J. Immunol. 154:685-693, 1995; Ruppert et al., Cell 74:929-937, 1993; Sidney et al., Immunol. Today 17:261-266, 1996; Sette and Sidney, Curr. Opin. in Immunol. 10:478482, 1998). Secondary anchor residues that characterize the A*0201 motif have additionally been defined (see, e.g., Ruppert et al., Cell 74:929-937, 1993). These secondary anchors are shown in Table 3. Peptide binding to HLA-A*0201 molecules can be modulated by substitutions at primary and/or secondary anchor positions, preferably choosing respective residues specified for the motif.

HLA-A24 Motif

The HLA-A24 motif is characterized by the presence in peptide ligands of Y, F, W, or M as a primary anchor residue in position 2, and F, L, I, or W as a primary anchor residue at the C-terminal position of the epitope (see, e.g., Kondo et al., J. Immunol. 155:4307-4312, 1995; and Kubo et al., J. Immunol. 152:3913-3924, 1994).

Motifs Indicative of Class II HTL Inducing Peptide Epitopes

The primary and secondary anchor residues of the HLA class II peptide epitope supermotifs and motifs are summarized in Table 4. Also see, U.S. Pat. Nos. 5,736,142, 5,679,640 and 6,413,935; co-pending applications entitled Alteration Of Immune Responses Using Pan DR Binding Peptides, U.S. Ser. No. 09/709,774, filed Nov. 8, 2000 and 09/707,738, filed Nov. 6, 2000; and PCT publication Nos. WO 95/07707 and WO 97/26784.

Enhancing Population Coverage of the Vaccine

As set forth in Tables 2 through 4, there are neumerous additional supermotifs and motifs in addition to the A2 supermotif and the A2.1-allel specific motif that presently are a focus of the present application. By inclusion of one or more epitopes from other motifs or supermotifs, enhanced population coverage for major global ethnicities can be obtained.

Immune Response-Stimulating Peptide Analogs

In general, CTL and HTL responses are not directed against all possible epitopes. Rather, they are restricted to a few “immunodominant” determinants (Zinkernagel, et al., Adv. Immunol 27:5159, 1979; Bennink, et al., J. Exp. Med. 168:19351939, 1988; Rawle, et al., J. Immunol. 146:3977-3984, 1991). It has been recognized that immunodominance (Benacerraf, et al., Science 175:273-279, 1972) could be explained by either the ability of a given epitope to selectively bind a particular HLA protein (determinant selection theory) (Vitiello, et al., J. Immunol. 131:1635, 1983); Rosenthal, et al., Nature 267:156-158, 1977), or to be selectively recognized by the existing TCR (T cell receptor) specificities (repertoire theory) (Klein, J., IMMUNOLOGY, TE SCIENCE OF SELFNONSELF DISCRIMINATION, John Wiley & Sons, New York, pp. 270-310, 1982). It has been demonstrated that additional factors, mostly linked to processing events, can also play a key role in dictating, beyond strict immunogenicity, which of the many potential determinants will be presented as immunodominant (Sercarz, et al., Annu. Rev. Immunol. 11:729-766, 1993).

The concept of dominance and subdominance is relevant to immunotherapy of both infectious diseases and malignancies. For example, in the course of chronic viral disease, recruitment of subdominant epitopes can be; important for successful clearance of the infection, especially if dominant CTL or HTL specificities have been inactivated by functional tolerance, suppression, mutation of viruses and other mechanisms (Franco, et al., Curr. Opin. Immunol. 7:524-531, 1995). In the case of cancer and tumor antigens, CTLs recognizing at least some of the highest binding affinity peptides might be functionally inactivated. Lower binding affinity peptides are preferentially recognized at these times, and may therefore be preferred in therapeutic or prophylactic anti-cancer vaccines.

In particular, it has been noted that a significant number of epitopes derived from known non-viral tumor associated antigens (TAA) bind HLA class I with intermediate affinity (IC₅₀ in the 50-500 nM range) rather than at high affinity (IC₅₀ of less than 50 nM).

For example, it has been found that 8 of 15 known TAA peptides recognized by tumor infiltrating lymphocytes (TIL) or CTL bound in the 50-500 nM range. (These data are in contrast with estimates that 90% of known viral antigens were bound by HLA class I molecules with IC₅₀ of 50 nM or less, while only approximately 10% bound in the 50-500 nM range (Sette, et al., J. Immunol., 153:558-5592, 1994). In the cancer setting this phenomenon is probably due to elimination or functional inhibition of the CTL recognizing several of the highest binding peptides, presumably because of T cell tolerization events.

Without intending to be bound by theory, it is believed that because T cells to dominant epitopes may have been clonally deleted, and selecting subdominant epitopes may allow existing T cells to be recruited, which will then lead to a therapeutic or prophylactic response. However, the binding of HLA molecules to subdominant epitopes is often less vigorous than to dominant ones.

Accordingly, there is a need to be able to modulate the binding affinity of particular immunogenic epitopes for one or more HLA molecules, to thereby modulate the immune response elicited by the peptide, for example to prepare analog peptides which elicit a more vigorous response. This ability to modulate both binding affinity and the resulting immune response in accordance with the present invention greatly enhances the usefulness of peptide epitope-based vaccines and therapeutic agents.

Although peptides with suitable cross-reactivity among all alleles of a superfamily are identified by the screening procedures described above, cross-reactivity is not always as complete as possible, and in certain cases procedures to increase cross-reactivity of peptides can be useful; moreover, such procedures can also be used to modify other properties of the peptides such as binding affinity or peptide stability. Having established the general rules that govern cross-reactivity of peptides for HLA alleles within a given motif or supermotif, modification (i.e., analoging) of the structure of peptides of particular interest in order to achieve broader (or otherwise modified) HLA binding capacity can be performed. More specifically, peptides that exhibit the broadest cross-reactivity patterns, can be produced in accordance with the teachings herein. The present concepts related to analog generation are set forth in greater detail in co-pending U.S. Ser. No. 09/226,775 filed 6 Jan. 1999.

In brief, the analoging strategy utilizes the motifs or supermotifs that correlate with binding to certain HLA molecules. Analog peptides can be created by substituting amino acid residues at primary anchor, secondary anchor, or at primary and secondary anchor positions. Generally, analogs are made for peptides that already bear a motif or supermotif. As noted herein, preferred primary and secondary anchor residues of supermotifs and motifs for MLA class I and HLA class II binding peptides are shown in Tables 3 and 4, respectively. For a number of the motifs or supermotifs in accordance with the invention, residues are defined which are deleterious to binding to allele-specific HLA molecules or members of HLA supertypes that bind the respective motif or supermotif (Tables 3 and 4). Accordingly, removal of such residues that are detrimental to binding can be performed in accordance with the present invention. For example, in the case of the A3 supertype, when all peptides that have such deleterious residues are removed from the population of peptides used in the analysis, the incidence of cross-reactivity increased from 22% to 37% (see, e.g., Sidney, J. et al., Hu. Immunol. 45:79, 1996).

Thus, one strategy to improve the cross-reactivity of peptides within a given supermotif is simply to delete one or more of the deleterious residues present within a peptide and substitute a small “neutral” residue such as Ala (that may not influence T cell recognition of the peptide). An enhanced likelihood of cross-reactivity is expected if, together with elimination of detrimental residues within a peptide, “preferred” residues associated with high affinity binding to an allele-specific HLA molecule or to multiple HLA molecules within a superfamily are inserted.

To ensure that an analog peptide, when used as a vaccine, actually elicits a CTL response to the native epitope in vivo (or, in the case of class II epitopes, elicits helper T cells that cross-react with the wild type peptides), the analog peptide may be used to induce T cells in vitro from individuals of the appropriate HLA allele. Thereafter, the immunized cells' capacity to lyse wild type peptide sensitized target cells is evaluated. Alternatively, evaluation of the cells' activity can be evaluated by monitoring UN release. Each of these cell monitoring strategies evaluate the recognition of the APC by the CTL. It will be desirable to use as antigen presenting cells, cells that have been either infected, or transfected with the appropriate genes, or, (generally only for class II epitopes, due to the different peptide processing pathway for HLA class II), cells that have been pulsed with whole protein antigens, to establish whether endogenously produced antigen is also recognized by the T cells induced by the analog peptide. It is to be noted that peptide/protein-pulsed dendritic cells can be used to present whole protein antigens for both HLA class I and class II.

Another embodiment of the invention is to create analogs of weak binding peptides, to thereby ensure adequate numbers of cellular binders. Class I binding peptides exhibiting binding affinities of 500-5000 nM, and carrying an acceptable but suboptimal primary anchor residue at one or both positions can be “fixed” by substituting preferred anchor residues in accordance with the respective supertype. The analog peptides can then be tested for binding and/or cross-binding capacity.

Another embodiment of the invention is to create analogs of peptides that are already cross-reactive binders and are vaccine candidates, but which bind weakly to one or more alleles of a supertype. If the cross-reactive binder carries a suboptimal residue (less preferred or deleterious) at a primary or secondary anchor position, the peptide can be analoged by substituting out a deleterious residue and replacing it with a preferred or less preferred one, or by substituting out a less preferred reside and replacing it with a preferred one. The analog peptide can then be tested for cross-binding capacity.

Another embodiment for generating effective peptide analogs involves the substitution of residues that have an adverse impact on peptide stability or solubility in, e.g., a liquid environment. This substitution may occur at any position of the peptide epitope. For example, a cysteine (C) can be substituted in favor of α-amino butyric acid. Due to its chemical nature, cysteine has the propensity to form disulfide bridges and sufficiently alter the peptide structurally so as to reduce binding capacity. Substituting α-amino butyric acid for C not only alleviates this problem, but actually improves binding and crossbinding capability in certain instances (see, e.g., the review by Sette et al., In: Persistent Viral Infections, Eds. R. Ahmed and I. Chen, John Wiley & Sons, England, 1999). Substitution of cysteine with α-amino butyric acid may occur at any residue of a peptide epitope, i.e. at either anchor or non-anchor positions.

Moreover, it has been shown that in sets of A*0201 motif-bearing peptides containing at least one preferred secondary anchor residue while avoiding the presence of any deleterious secondary anchor residues, 69% of the peptides will bind A*0201 with an IC₅₀ less than 500 nM (Ruppert, J. et al. Cell 74:929, 1993). The determination of what was a preferred or deleterious residue in Ruppert can be used to generate algorithms. Such algorithms are flexible in that cut-off scores may be adjusted to select sets of peptides with greater or lower predicted binding properties, as desired.

In accordance with the procedures described herein, tumor associated antigen peptide epitopes and analogs thereof that were found to bind HLA-A1, -A2, -A3, -A11, -A24, -B7 and -B44 allele-specific molecules and to members of the HLA-A2, -A11, -B7 and -B44 supertypes have been identified.

Furthermore, additional amino acids can be added to the termini of a peptide to provide for ease of linking peptides one to another, for coupling to a carrier support or larger peptide, for modifying the physical or chemical properties of the peptide or oligopeptide, or the like. Amino acids such as tyrosine, cysteine, lysine, glutamic or aspartic acid, or any naturally occuring or any non-naturally occuring amino acid residues, can be introduced at the C- and/or N-terminus of the peptide or oligopeptide, particularly class I peptides. It is to be noted that modification at the carboxyl terminus of a CTL epitope may, in some cases, alter binding characteristics of the peptide. In addition, the peptide or oligopeptide sequences can differ from the natural sequence by being modified by terminal-NH₂ acylation, e.g., by alkanoyl (C₁-C₂₀) or thioglycolyl acetylation, terminal-carboxylamidation, e.g., ammonia, methylamine, etc. In some instances these modifications may provide sites for linking to a support or other molecule.

Assays to Detect T-Cell Responses

Once HLA binding peptides are identified, they can be tested for the ability to elicit a T-cell response. The preparation and evaluation of motif-bearing peptides are described, e.g., in PCT publications WO 94/20127 and WO 94/03205. Briefly, peptides comprising epitopes from a particular antigen are synthesized and tested for their ability to bind to relevant HLA proteins. These assays may involve evaluation of peptide binding to purified HLA class I molecules in relation to the binding of a radioiodinated reference peptide. Alternatively, cells expressing empty class I molecules (i.e. cell surface HLA molecules that lack any bound peptide) may be evaluated for peptide binding by immunofluorescent staining and flow microfluorimetry. Other assays that may be used to evaluate peptide binding include peptide-dependent class I assembly assays and/or the inhibition of CTL recognition by peptide competition. Those peptides that bind to an HLA class I molecule, typically with an affinity of 500 nM or less, are further evaluated for their ability to serve as targets for CTLs derived from infected or immunized individuals, as well as for their capacity to induce primary in vitro or in vivo CTL responses that can give rise to CTL populations capable of reacting with selected target cells associated with pathology.

Analogous assays are used for evaluation of HLA class II binding peptides. HLA class II motif-bearing peptides that are shown to bind, typically at an affinity of 1000 nM or less, are further evaluated for the ability to stimulate HTL responses.

Conventional assays utilized to detect T cell responses include proliferation assays, lymphokine secretion assays, direct cytotoxicity assays, and limiting dilution assays. For example, antigen-presenting cells that have been incubated with a peptide can be assayed for the ability to induce CTL responses in responder cell populations. Antigen-presenting cells can be normal cells such as peripheral blood mononuclear cells or dendritic cells. Alternatively, mutant, non-human mammalian cell lines that have been transfected with a human class I MHC gene, and that are deficient in their ability to load class I molecules with internally processed peptides, are used to evaluate the capacity of the peptide to induce in vitro primary CTL responses. Peripheral blood mononuclear cells (PBMCs) can be used as the source of CTL precursors. Antigen presenting cells are incubated with peptide, after which the peptide-loaded antigen-presenting cells are then incubated with the responder cell population under optimized culture conditions. Positive CTL activation can be determined by assaying the culture for the presence of CTLs that lyse radio-labeled target cells, either specific peptide-pulsed targets or target cells that express endogenously processed antigen from which the specific peptide was derived. Alternatively, the presence of epitope-specific CTLs can be determined by IFNγ in situ ELISA.

In an embodiment of the invention, directed to diagnostics, a method has been devised which allows direct quantification of antigen-specific T cells by staining with fluorescein-labelled HLA tetrameric complexes (Altman, J. D. et al., Proc. Natl. Acad. Sci. USA 90:10330, 1993; Altman, J. D. et al., Science 274:94, 1996). Other options include staining for intracellular lympholines, and interferon release assays or ELISPOT assays. Tetramer staining, intracellular lymphokine staining and ELISPOT assays all appear to be at least 10-fold more sensitive than more conventional assays (Lalvani, A. et al, J. Exp. Med. 186:859, 1997; Dunbar, P. R. et al., Curr. Biol. 8:413, 1998; Murali-Krishna, K. et al., Immunity 8:177, 1998). Additionally, DimerX technology can be used as a means of quantitation (see, e.g., Science 274:94-99 (1996) and Proc. Natl. Acad. Sci. 95:7568-73 (1998)).

HTL activation may also be assessed using techniques known to those in the art, such as T cell proliferation or lympholine secretion (see, e.g. Alexander et al., Immunity 1:751-761, 1994).

Alternatively, immunization of HLA transgenic mice can be used to determine immunogenicity of peptide epitopes. Several transgenic mouse strains, e.g., mice with human A2.1, A11(which can additionally be used to analyze HLA-A3 epitopes), and B7 alleles have been characterized. Other transgenic mice strains (e.g., transgenic mice for HLA-A1 and A24) are being developed. Moreover, HLA-DR1 and HLA-DR3 mouse models have been developed. In accordance with principles in the art, additional transgenic mouse models with other HLA alleles are generated as necessary.

Such mice can be immunized with peptides emulsified in Incomplete Freund's Adjuvant; thereafter any resulting T cells can be tested for their capacity to recognize target cells that have been peptide-pulsed or transfected with genes encoding the peptide of interest. CTL responses can be analyzed using cytotoxicity assays described above. Similarly, HTL responses can be analyzed using, e.g., T cell proliferation or lympholine secretion assays.

Enhancing Population Coverage of the Vaccine

As set forth in Tables 2 through 4, there are numerous additional supermotifs and motifs in addition to the A3, B7 and B44 supermotifs and motifs and A1, A2, A24 and B44 motifs that presently are a focus of the present application. By inclusion of one or more epitopes from other motifs or supermotifs, enhanced population coverage for major global ethnicities can be obtained. (See Tables 11, 13a, 13b, and 32).

Minigenes

A number of different approaches are available which allow simultaneous delivery of multiple epitopes. Nucleic acids encoding multiple epitopes are a useful embodiment of the invention; discrete peptide epitopes or polyepitopic peptides can be encoded. The epitopes to be included in a minigene are preferably selected according to the guidelines set forth in the previous section. Examples of amino acid sequences that can be included in a minigene include: HLA class I epitopes, HLA class II epitopes, a ubiquitination signal sequence, and/or a targeting sequence such as an endoplasmic reticulum (ER) signal sequence to facilitate movement of the resulting peptide into the endoplasmic reticulum.

The use of multi-epitope minigenes is also described in, e.g. co-pending applications U.S. Ser. Nos. 09/311,784, 09/894,018, 60/419,973, 60/415,463; Ishioka et al., J. Immunol. 162:3915-3925, 1999; An, L. and Whitton, J. L., J. Virol. 71:2292, 1997; Thomson, S. A. et al., J. Immunol. 157:822, 1996; Whitton, J. L. et al., J. Virol. 67:348, 1993; Hanke, R. et al., Vaccine 16:426, 1998. For example, a multi-epitope DNA plasmid encoding nine dominant HLA-A*0201- and A11-restricted CTL epitopes derived from the polymerase, envelope, and core proteins of HBV and human immunodeficiency virus (HIV), a PADRE® universal helper T cell (HTL) epitope, and an endoplasmic reticulum-translocating signal sequence has been engineered. Immunization of HLA transgenic mice with this plasmid construct resulted in strong CTL induction responses against the nine CTL epitopes tested. This CTL response was similar to that observed with a lipopeptide of known immunogenicity in humans, and significantly greater than immunization using peptides in oil-based adjuvants. Moreover, the immunogenicity of DNA-encoded epitopes in vitro was also correlated with the in vitro responses of specific CTL lines against target cells transfected with the DNA plasmid. These data show that the minigene served: 1.) to generate a CTL response and 2.) to generate CTLs that recognized cells expressing the encoded epitopes. A similar approach can be used to develop minigenes encoding TAA epitopes.

For example, to create a DNA sequence encoding the selected epitopes (minigene) for expression in human cells, the amino acid sequences of the epitopes may be reverse translated. A human codon usage table can be used to guide the codon choice for each amino acid. These epitope-encoding DNA sequences may be directly adjoined, so that when translated, a continuous peptide sequence is created. However, to optimize expression and/or immunogenicity, additional elements can be incorporated into the minigene design such as spacer amino acid residues between epitopes. HLA presentation of CTL and HTL epitopes may be improved by including synthetic (e.g. poly-alanine) or naturally-occurring flanlkng sequences adjacent to the CTL or HTL epitopes; these larger peptides comprising the epitope(s) are within the scope of the invention. In one embodiment, spacer amino acid residues between one or more CTL and/or HTL epitopes are designed so as to minimize junctional epitopes that may result from the juxtaposition of 2 CTL and/or HTL epitopes.

The minigene sequence may be converted to DNA by assembling oligonucleotides that encode the plus and minus strands of the minigene. Overlapping oligonucleotides (30-100 bases long) may be synthesized, phosphorylated, purified and annealed under appropriate conditions using well known techniques. The ends of the oligonucleotides can be joined, for example, using T4 DNA ligase. This synthetic minigene, encoding the epitope peptide, can then be cloned into a desired expression vector.

Standard regulatory sequences well known to those of skill in the art are preferably included in the vector to ensure expression in the target cells. Several vector elements are desirable: a promoter with a downstream cloning site for minigene insertion; a polyadenylation signal for efficient transcription termination; an E. coli origin of replication; and an E. coli selectable marker (e.g. ampicillin or kanamycin resistance). Numerous promoters can be used for this purpose, e.g., the human cytomegalovirus (hCMV) CMV-IE promoter. See, e.g., U.S. Pat. Nos. 5,580,859 and 5,589,466 for other suitable promoter sequences.

Optimized peptide expression and immunogenicity can be achieved by certain modifications to a minigene construct. For example, in some cases introns facilitate efficient gene expression, thus one or more synthetic or naturally-occurring introns can be incorporated into the transcribed region of the minigene. The inclusion of mRNA stabilization sequences and sequences for replication in mammalian cells may also be considered for increasing minigene expression.

Once an expression vector is selected, the minigene is cloned into the polylinker region downstream of the promoter. This plasmid is transformed into an appropriate bacterial strain, and DNA is prepared using standard techniques. The orientation and DNA sequence of the minigene, as well as all other elements included in the vector, are confirmed using restriction mapping, PCR and/or DNA sequence analysis. Bacterial cells harboring the correct plasmid can be stored as cell banks.

In addition, immunostimulatory sequences (ISSs or CpGs) appear to play a role in the immunogenicity of DNA vaccines. These sequences may be included in the vector, outside the minigene coding sequence to enhance immunogenicity.

In some embodiments, a bi-cistronic expression vector which allows production of both the minigene-encoded epitopes and a second protein (e.g., one that modulates immunogenicity) can be used. Examples of proteins or polypeptides that, if co-expressed with epitopes, can enhance an immune response include cytokines (e.g., IL-2, IL-12, GM-CSF), cytokine-inducing molecules (e.g., LeIF), costimulatory molecules, or pan-DR binding proteins (PADRE®, Epimmune, San Diego, Calif.). Helper T cell (HTL) epitopes such as PADRE® molecules can be joined to intracellular targeting signals and expressed separately from expressed CTL epitopes. This can be done in order to direct HTL epitopes to a cell compartment different than that of the CTL epitopes, one that provides for more efficient entry of HTL epitopes into the “LA class II pathway, thereby improving HTL induction. In contrast to HTL or CTL induction, specifically decreasing the immune response by co-expression of immunosuppressive molecules (e.g. TGF-β) may be beneficial in certain diseases.

Therapeutic quantities of plasmid DNA can be produced for example, by fermentation in E. coli, followed by purification. Aliquots from the working cell bank are used to inoculate growth medium, and are grown to saturation in shaker flasks or a bioreactor according to well known techniques. Plasmid DNA is purified using standard bioseparation technologies such as solid phase anion-exchange resins available, e.g., from QIAGEN, Inc. (Valencia, Calif.). If required, supercoiled DNA can be isolated from the open circular and linear forms using gel electrophoresis or other methods.

Purified plasmid DNA can be prepared for injection using a variety of formulations. The simplest of these is reconstitution of lyophilized DNA in sterile phosphate-buffer saline (PBS). This approach, known as “naked DNA,” is currently being used for intramuscular (IM) administration in clinical trials. To maximize the immunotherapeutic effects of minigene vaccines, alternative methods of formulating purified plasmid DNA may be used. A variety of such methods have been described, and new techniques may become available. Cationic lipids, glycolipids, and fusogenic liposomes can also be used in the formulation (see, e.g., WO 93/24640; Mannino & Gould-Fogerite, BioTeclniques 6(7): 682 (1988); U.S. Pat. No. 5,279,833; WO 91/06309; and Felgner, et al., Proc. Nat'l Acad Sci. USA 84:7413 (1987). In addition, peptides and compounds referred to collectively as protective, interactive, non-condensing compounds (PINC) can also be complexed to purified plasmid DNA to influence variables such as stability, intramuscular dispersion, or trafficking to specific organs or cell types.

Known methods in the art can be used to enhance delivery and uptake of a polynucleotide in vivo. For example, the polynucleotide can be complexed to polyvinylpyrrolidone (PVP), to prolong the localized bioavailability of the polynucleotide, thereby enhancing uptake of the polynucleotide by the organisum (see e.g., U.S. Pat. No. 6,040,295; EP 0 465 529; WO 98/17814). PVP is a polyamide that is known to form complexes with a wide variety of substances, and is chemically and physiologically inert.

Target cell sensitization can be used as a functional assay of the expression and HLA class I presentation of minigene-encoded epitopes. For example, the plasmid DNA is introduced into a mammalian cell line that is a suitable target for standard CTL chromium release assays. The transfection method used will be dependent on the final formulation, electroporation can be used for “naked” DNA, whereas cationic lipids or DNA:PVP compositions allow direct in vitro transfection. A plasmid expressing green fluorescent protein (GFP) can be co-transfected to allow enrichment of transfected cells using fluorescence activated cell sorting (FACS). The transfected cells are then chromium-51 (⁵¹Cr) labeled and used as targets for epitope-specific CTLs. Cytolysis of the target cells, detected by ⁵¹Cr release, indicates both the production and HLA presentation of, minigene-encoded CTL epitopes. Expression of HTL epitopes may be evaluated in an analogous manner using assays to assess HTL activity.

In vivo immunogenicity is a second approach for functional testing of minigene DNA formulations. Transgenic mice expressing appropriate human HLA proteins are immunized with the DNA product. The dose and route of administration are formulation dependent (e.g., IM for DNA in PBS, intraperitoneal (IP) for lipid-complexed DNA). Eleven to twenty-one days after immunization, splenocytes are harvested and restimulated for one week in the presence of peptides encoding each epitope being tested. Thereafter, for CTLs, standard assays are conducted to determine if there is cytolysis of peptide-loaded, ⁵¹Cr-labeled target cells. Once again, lysis of target cells that were exposed to epitopes corresponding to those in the minigene, demonstrates DNA vaccine function and induction of CTLs. Immunogenicity of HTL epitopes is evaluated in transgenic mice in an analogous manner.

Alternatively, the nucleic acids can be administered using ballistic delivery as described, for instance, in U.S. Pat. No. 5,204,253. Using this technique, particles comprised solely of DNA are administered. In a further alternative embodiment for ballistic delivery, DNA can be adhered to particles, such as gold particles.

Vaccine Compositions

Vaccines that contain an immunologically effective amount of one or more peptides or polynucleotides of the invention are a further embodiment of the invention. The peptides can be delivered by various means or formulations, all collectively referred to as “vaccine” compositions. Such vaccine compositions, and/or modes of administration, can include, for example, naked DNA, DNA formulated with PVP, DNA in cationic lipid formulations; lipopeptides (e.g., Vitiello, A. et al., J. Clin. Invest. 95:341, 1995), DNA or peptides, encapsulated e.g. in poly(DL-lactide-co-glycolide) (“PLG”) microspheres (see, e.g., Eldridge, et al., Molec. Immunol. 28:287-294, 1991: Alonso et al., Vaccine 12:299-306, 1994; Jones et al., Vaccine 13:675-681, 1995); peptide compositions contained in immune stimulating complexes (ISCOMS) (see, e.g., Takahashi et al., Nature 344:873-875, 1990; Hu et al., Clin Exp Immunol. 113:235-243, 1998); multiple antigen peptide systems (MAPs) (see e.g., Tam, J. P., Proc. Natl. Acad. Sci. U.S.A. 85:5409-5413, 1988; Tam, J. P., J. Immunol. Methods 196:17-32, 1996); viral, bacterial, or, fungal delivery vectors (Perkus, M. E. et al., In: Concepts in vaccine development, Kaufmann, S. H. E., ed., p. 379, 1996; Chakrabarti, S. et al., Nature 320:535, 1986; Hu, S. L. et al., Nature 320:537, 1986; Kieny, M.-P. et al., AIDS Bio/Technology 4:790, 1986; Top, F. H. et al., J. Infect. Dis. 124:148, 1971; Chanda, P. K. et al., Virology 175:535, 1990); particles of viral or synthetic origin (e.g. Kofler, N. et al., J. Immunol. Methods. 192:25, 1996; Eldridge, J. H. et al., Sem. Hematol. 30:16, 1993; Falo, L. D., Jr. et al., Nature Med. 7:649, 1995); adjuvants (e.g. incomplete freund's advjuvant) (Warren, H. S., Vogel, F. R., and Chedid, L. A. Annu. Rev. Immunol. 4:369, 1986; Gupta, R. K. et al., Vaccine 11:293, 1993); liposomes (Reddy, R et al., J. Immunol. 148:1585, 1992; Rock, K. L., Immunol. Today 17:131, 1996); or, particle-absorbed DNA (Ulmer, J. B. et al., Science 259:1745, 1993; Robinson, H. L., Hunt, L. A., and Webster, R. G., ii Vaccine 11:957, 1993; Shiver, J. W. et al., In: Concepts in vaccine development, Kaufmann, S. H. E., ed., p. 423, 1996; Cease, K. B., and Berzofsky, J. A., Annu. Rev. Immunol. 12:923, 1994 and Eldridge, J. H. et al., Sem. Hematol. 30:16, 1993), etc. Toxin-targeted delivery technologies, also known as receptor mediated targeting, such as those of Avant Immunotherapeutics, Inc. (Needham, Mass.) or attached to a stress protein, e.g., HSP 96 (Stressgen Biotechnologies Corp., Victoria, BC, Canada) can also be used.

Vaccines of the invention comprise nucleic acid mediated modalities. DNA or RNA encoding one or more of the peptides of the invention can be administered to a patient. This approach is described, for instance, in Wolff et. al., Science 247:1465 (1990) as well as U.S. Pat. Nos. 5,580,859; 5,589,466; 5,804,566; 5,739,118; 5,736,524; 5,679,647; and, WO 98/04720. Examples of DNA-based delivery technologies include “naked DNA”, facilitated (bupivicaine, polymers (e.g., PVP), peptide-mediated) delivery, cationic lipid complexes, and particle-mediated (“gene gun”) or pressure-mediated delivery (see, e.g., U.S. Pat. No. 5,922,687). Accordingly, peptide vaccines of the invention can be expressed by viral or bacterial vectors. Examples of expression vectors include attenuated viral hosts, such as vaccinia or fowlpox. For example, vaccinia virus is used as a vector to express nucleotide sequences that encode the peptides of the invention (e.g., MVA). Upon introduction into an acutely or chronically infected host or into a non-infected host, the recombinant vaccinia virus expresses the immunogenic peptide, and thereby elicits an immune response. Vaccinia vectors and methods useful in immunization protocols are described in, e.g., U.S. Pat. No. 4,722,848. Another vector is BCG (Bacille Calmette Guerin). BCG vectors are described in Stover et al., Nature 351:456-460 (1991). A wide variety of other vectors useful for therapeutic administration or immunization of the peptides of the invention, e.g. adeno and adeno-associated virus vectors, alpha virus vectors, retroviral vectors, Salmonella typhi vectors, detoxified anthrax toxin vectors, and the like, are apparent to those skilled in the art from the description herein.

Furthermore, vaccines in accordance with the invention can comprise one or more peptides of the invention. Accordingly, a peptide can be present in a vaccine individually; alternatively, the peptide can exist as a homopolymer comprising multiple copies of the same peptide, or as a heteropolymer of various peptides. Polymers have the advantage of increased probability for immunological reaction and, where different peptide epitopes are used to make up the polymer, the ability to induce antibodies and/or T cells that react with different antigenic determinants of the antigen targeted for an immune response. The composition may be a naturally occurring region of an antigen or can be prepared, e.g., recombinantly or by chemical synthesis.

Carriers that can be used with vaccines of the invention are well known in the art, and include, e.g., thyroglobulin, albumins such as human serum albumin, tetanus toxoid, polyamino acids such as poly L-lysine, poly L-glutanic acid, influenza virus proteins, hepatitis B virus core protein, and the like. The vaccines can contain a physiologically tolerable diluent such as water, or a saline solution, preferably phosphate buffered saline. Generally, the vaccines also include an adjuvant. Adjuvants such as incomplete Freund's adjuvant, aluminum phosphate, aluminum hydroxide, or alum are examples of materials well known in the art. Additionally, as disclosed herein, CTL responses can be primed by conjugating peptides of the invention to lipids, such as tripalmitoyl-S-glyceryl-cysteinyl-seryl-serine (P₃CSS).

Upon immunization with a peptide composition in accordance with the invention, via injection (e.g., SC, ID, IM), aerosol, oral, transdermal, transmucosal, intrapleural, intrathecal, or other suitable routes, the immune system of the host responds to the vaccine by producing antibodies, CTLs and/or HTLs specific for the desired antigen. Consequently, the host becomes at least partially immune to subsequent exposure to the TAA, or at least partially resistant to further development of TAA-bearing cells and thereby derives a prophylactic or therapeutic benefit.

In certain embodiments, components that induce T cell responses are combined with components that induce antibody responses to the target antigen of interest. A preferred embodiment of such a composition comprises class I and class II epitopes in accordance with the invention. Alternatively, a composition comprises a class I and/or class II epitope in accordance with the invention, along with a PADRE® molecule (Epimmune, San Diego, Calif.).

Vaccines of the invention can comprise antigen presenting cells, such as dendritic cells, as a vehicle to present peptides of the invention. For example, dendritic cells are transfected, e.g., with a minigene construct in accordance with the invention, in order to elicit immune responses. Minigenes are discussed in greater detail in a following section. Vaccine compositions can be created in vitro, following dendritic cell mobilization and harvesting, whereby loading of dendritic cells occurs in vitro.

The vaccine compositions of the invention may also be used in combination with antiviral drugs such as interferon-α, or immune adjuvants such as IL-12, GM-CSF, etc.

Preferably, the following principles are utilized when selecting epitope(s) and/or analogs for inclusion in a vaccine, either peptide-based or nucleic acid-based formulations. Exemplary epitopes and analogs that may be utilized in a vaccine to treat or prevent TAA-associated disease are set out in Table 6. Each of the following principles can be balanced in order to make the selection. When multiple epitopes are to be used in a vaccine, the epitopes may be, but need not be, contiguous in sequence in the native antigen from which the epitopes are derived. Such multiple epitotes can refer to the order of epitopes within a peptide, or to the selection of epitopes that come from the same reagion, for use in either individual peptides or in a multi-epitopic peptide.

1.) Epitopes and/or analogs are selected which, upon administration, mimic immune responses that have been observed to be correlated with prevention or clearance of TAA-expressing tumors. For HLA Class L this generally includes 3-4 epitopes and/or analogs from at least one TAA.

2.) Epitopes and/or analogs are selected that have the requisite binding affinity established to be correlated with immunogenicity: for HLA Class I an IC₅₀ of 500 nM or less, or for Class II an IC₅₀ of 1000 nM or less. For HLA Class I it is presently preferred to select a peptide having an IC₅₀ of 200 nM or less, as this is believed to better correlate not only to induction of an immune response, but to in vitro tumor cell killing as well. For HLA A1 and A24, it is especially preferred to select a peptide having an IC₅₀ of 100 nM or less.

3.) Supermotif bearing-epitopes and/or analogs, or a sufficient array of allele-specific motif-bearing epitopes and/or analogs, are selected to give broad population coverage. In general, it is preferable to have at least 80% population coverage. A Monte Carlo analysis, a statistical evaluation known in the art, can be employed to assess the breadth of population coverage.

4.) For cancer-related antigens, it can be preferable to select analogs instead of or in addition to epitopes, because the patient may have developed tolerance to the native epitope.

5.) Of particular relevance are “nested epitopes.” Nested epitopes occur where at least two epitopes overlap in a given peptide sequence. For example, a nested epitope can be a fragment of an antigen from a region that contains multiple epitopes that are overleapping, or one epitope that is completely encompassed by another, e.g., A2 peptides MAGE3.159 and MAGE3.160 are nested. A peptide comprising “transcendent nested epitopes” is a peptide that has both HLA class, I and HLA class II epitopes in it. When providing nested epitopes, it is preferable to provide a sequence that has the greatest number of epitopes per provided sequence. Preferably, one avoids providing a peptide that is any longer than the amino terminus of the amino terminal epitope and the carboxyl terminus of the carboxyl terminal epitope in the peptide. When providing a sequence comprising nested epitopes, it is important to evaluate the sequence in order to insure that it does not have pathological or other deleterious biological properties; this is particularly relevant for vaccines directed to infectious organisms.

6.) If a protein with multiple epitopes or a polynucleotide (e.g., minigene) is created, an objective is to generate the smallest peptide that encompasses the epitopes of interest. This principle is similar, if not the same as that employed when selecting a peptide comprising nested epitopes. However, with an artificial peptide comprising multipe epitopes, the size minimization objective is balanced against the need to integrate any spacer sequences between epitopes in the polyepitopic protein. Spacer amino acid residues can be introduced to avoid junctional epitopes (an epitope recognized by the immune system, not present in the target antigen, and only created by the man-made juxtaposition of epitopes), or to facilitate cleavage between epitopes and thereby enhance epitope presentation. Junctional epitopes are generally to be avoided because the recipient may generate an immune response to that non-native epitope. Of particular concern is a junctional epitope that is a “dominant epitope.” A dominant epitope may lead to such a zealous response that immune responses to other epitopes are diminished or suppressed.

The principles are the same, except junctional epitopes applies to the sequences surrounding the epitope. One must also take care with other sequences in construct to avoid immune response.

T Cell Priming Materials

In some embodiments it may be desirable to include in the pharmaceutical compositions of the invention at least one component which primes cytotoxic T lymphocytes. Lipids have been identified as agents capable of facilitating the priming in vitro CTL response against viral antigens. For example, palmitic acid residues can be attached to the δ- and α-amino groups of a lysine residue and then linked to an immunogenic peptide. One or more linking moieties can be used such as Gly, Gly-Gly-, Ser, Ser-Ser, or the like. The lipidated peptide can then be administered directly in a micelle or particle, incorporated into a liposome, or emulsified in an adjuvant, e.g., incomplete Freund's adjuvant. A preferred immunogenic composition comprises palmitic acid attached to ε- and α-amino groups of Lys via a linking moiety, e.g., Ser-Ser, added to the amino terminus of an immunogenic peptide.

In another embodiment of lipid-facilitated priming of CTL responses, E. coli lipoproteins, such as tripalmitoyl-S-glyceryl-cysteinyl-seryl-serine (P₃CSS) can be used to prime CTL when covalently attached to an appropriate peptide. (See, e.g., Deres, et al., Nature 342:561, 1989). Thus, peptides of the invention can be coupled to P₃CSS, and the lipopeptide administered to an individual to specifically prime a CTL response to the target antigen. Moreover, because the induction of neutralizing antibodies can also be primed with P₃CSS-conjugated epitopes, two such compositions can be combined to elicit both humoral and cell-mediated responses.

Dendritic Cells Pulsed with CTL and/or HTL Peptides

An embodiment of a vaccine composition in accordance with the invention comprises ex vivo administration of a cocktail of epitope-bearing peptides to PBMC, or isolated DC therefrom, from the patient's blood. A pharmaceutical to facilitate harvesting of DC can be used, such as Progenipoietin™ (Monsanto, St. Louis, Mo.) or GM-CSF/IL-4. After pulsing the DC with peptides and prior to reinfusion into patients, the DC are washed to remove unbound peptides. In this embodiment, a vaccine comprises peptide-pulsed DCs which present the pulsed peptide epitopes in HLA molecules on their surfaces.

The DC can be pulsed ex vivo with a cocktail of peptides, some of which stimulate CTL responses to one or more antigens of interest, e.g. tumor associated antigens (TAA) such as HER2/neu, p53, MAGE 2, MAGE3, and/or carcinoembryonic antigen (CEA). Collectively, these TAA are associated with breast, colon and lung cancers. Optionally, a helper T cell (HTL) peptide such as PADRE®, can be included to facilitate the CTL response. Thus, a vaccine in accordance with the invention comprising epitopes from HER2/neu, p53, MAGE 2, MAGE3, and carcinoembryonic antigen (CEA) is used to treat minimal or residual disease in patients with malignancies such as breast, colon, lung or ovarian cancer; any malignancies that bear any of these TAAs can also be treated with the vaccine. A TAA vaccine can be used following debulking procedures such as surgery, radiation therapy or chemotherapy, whereupon the vaccine provides the benefit of increasing disease free survival and overall survival in the recipients.

Thus, in preferred embodiments, a vaccine of the invention is a product that treats a majority of patients across a number of different tumor types. A vaccine comprising a plurality of epitopes, preferably supermotif-bearing epitopes, offers such an advantage.

Diagnostic and Prognostic Uses

In one embodiment of the invention, HLA class I and class II binding peptides can be used as reagents to evaluate an immune response. Preferably, the following principles are utilized when selecting an epitope(s) and/or analog(s) for diagnostic, prognostic and similar uses. Potential principles include having the binding affinities described earlier, and/or matching the HLA-motif/supermotif of a peptide with the HLA-type of a patient.

The evaluated immune response can be induced by any immunogen. For example, the immunogen may result in the production of antigen-specific CTLs or HTLs that recognize the peptide epitope(s) employed as the reagent. Thus, a peptide of the invention may or may not be used as the immunogen. Assay systems that can be used for such analyses include tetramer-based protocols (e.g., DimerX technology (see, e.g., Science 274:94-99 (1996) and Proc. Natl. Acad. Sci. 95:7568-73 (1998)), staining for intracellular lymphokines, interferon release assays, or ELISPOT assays.

For example, following exposure to a putative immunogen, a peptide of the invention can be used in a tetramer staining assay to assess peripheral blood mononuclear cells for the presence of any antigen-specific CTLs. The HLA-tetrameric complex is used to directly visualize antigen-specific CTLs and thereby determine the frequency of such antigen-specific CTLs in a sample of peripheral blood mononuclear cells (see, e.g., Ogg et al., Science 279:2103-2106, 1998; and Altman et al., Science 174:94-96, 1996).

A tetramer reagent comprising a peptide of the invention is generated as follows: A peptide that binds to an HLA molecule is refolded in the presence of the corresponding HLA heavy chain and β₂-microglobulin to generate a trimolecular complex. The complex is biotinylated at the carboxyl terminal end of the HLA heavy chain, at a site that was previously engineered into the protein. Tetramer formation is then induced by adding streptavidin. When fluorescently labeled streptavidin is used, the tetrameric complex is used to stain antigen-specific cells. The labeled cells are then readily identified, e.g., by flow cytometry. Such procedures are used for diagnostic or prognostic purposes; the cells identified by the procedure can be used for therapeutic purposes.

Peptides of the invention are also used as reagents to evaluate immune recall responses. (see, e.g., Bertoni et al., J. Clin. Invest. 100:503-513, 1997 and Penna et al., J. Exp. Med. 174:1565-1570, 1991.) For example, a PBMC sample from an individual expressing a disease-associated antigen (e.g. a tumor-associated antigen such as CEA, p53, MAGE2/3,HER2neu, or an organism associated with neoplasia such as HPV or HSV) can be analyzed for the presence of antigen-specific CTLs or HTLs using specific peptides. A blood sample containing mononuclear cells may be evaluated by cultivating the PBMCs and stimulating the cells with a peptide of the invention. After an appropriate cultivation period, the expanded cell population may be analyzed, for example, for CTL or for HTL activity.

Thus, the peptides can be used to evaluate the efficacy of a vaccine. PBMCs obtained from a patient vaccinated with an immunogen may be analyzed by methods such as those described herein. The patient is HLA typed, and peptide epitopes that are bound by the HLA molecule(s) present in that patient are selected for analysis. The immunogenicity of the vaccine is indicated by the presence of CTLs and/or HTLs directed to epitopes present in the vaccine.

The peptides of the invention may also be used to make antibodies, using techniques well known in the art (see, e.g. CURRENT PROTOCOLS IN IMMUNOLOGY, Wiley/Greene, NY; and Antibodies A Laboratory Manual Harlow, Harlow and Lane, Cold Spring Harbor Laboratory Press, 1989). Such antibodies are useful as reagents to determine the presence of disease-associated antigens. Antibodies in this category include those that recognize a peptide when bound by an HLA molecule, i.e., antibodies that bind to a peptide-MHC complex.

Administration for Therapeutic or Prophylactic Purposes

The peptides and polynucleotides of the present invention, including compositions thereof, are useful for administration to mammals, particularly humans, to treat and/or prevent disease. In one embodiment, peptides, polynucleotides, or vaccine compositions (peptide or nucleic acid) of the invention are administered to a patient who has a malignancy associated with expression of one or more TAAs, or to an individual susceptible to, or otherwise at risk for developing TAA-related disease. Upon administration an immune response is elicited against the TAAs, thereby enhancing the patient's own immune response capabilities. In therapeutic applications, peptide and/or nucleic acid compositions are administered to a patient in an amount sufficient to elicit an effective immune response to the TAA-expressing cells and to thereby cure, arrest or slow symptoms and/or complications. An amount adequate to accomplish this is defined as “therapeutically effective dose.” Amounts effective for this use will depend on, e.g., the particular composition administered, the manner of administration, the stage and severity of the disease being treated, the weight and general state of health of the patient, and the judgment of the prescribing physician.

The vaccine compositions of the invention can be used purely as prophylactic agents. Generally the dosage for an initial prophylactic immunization generally occurs in a unit dosage range where the lower value is about 1, 5, 50, 500, or 1000 μg of peptide and the higher value is about 10,000; 20,000; 30,000; or 50,000 μg of peptide. Dosage values for a human typically range from about 500 μg to about 50,000 μg of peptide per 70 kilogram patient. This is followed by boosting dosages of between about 1.0 μg to about 50,000 μg of peptide, administered at defined intervals from about four weeks to six months after the initial administration of vaccine. The immunogenicity of the vaccine may be assessed by measuring the specific activity of CTL and HTL obtained from a sample of the patient's blood.

As noted above, peptides comprising CTL and/or HTL epitopes of the invention induce immune responses when presented by HLA molecules and contacted with a CTL or HTL specific for an epitope comprised by the peptide. The manner in which the peptide is contacted with the CTL or HTL is not critical to the invention. For instance, the peptide can be contacted with the CTL or HTL either in vitro or in vivo. If the contacting occurs in vivo, peptide can be administered directly, or in other forms/vehicles, e.g. DNA vectors encoding one or more peptides, viral vectors encoding the peptide(s), liposomes, antigen presenting cells such as dendritic cells, and the like.

Accordingly, for pharmaceutical compositions of the invention in the form of peptides or polypeptides, the peptides or polypeptides can be administered directly. Alternatively, the peptide/polypeptides can be administered indirectly presented on APCs, or as DNA encoding them. Furthermore, the peptides or DNA encoding them can be administered individually or as fusions of one or more peptide sequences.

For therapeutic use, administration should generally begin at the first diagnosis of TAA-related disease. This is followed by boosting doses at least until symptoms are substantially abated and for a period thereafter. In chronic disease states, loading doses followed by boosting doses may be required.

The dosage for an initial therapeutic immunization generally occurs in a unit dosage range where the lower value is about 1, 5, 50, 500, or 1,000 μg of peptide and the higher value is about 10,000; 20,000; 30,000; or 50,000 μg of peptide. Dosage values for a human typically range from about 500 μg to about 50,000 μg of peptide per 70 kilogram patient. Boosting dosages of between about 1.0 μg to about 50,000 μg of peptide, administered pursuant to a boosting regimen over weeks to months, can be administered depending upon the patient's response and condition. Patient response can be determined by measuring the specific activity of CTL and HTL obtained from the patient's blood.

In certain embodiments, peptides and compositions of the present invention are used in serious disease states. In such cases, as a result of the minimal amounts of extraneous substances and the relative nontoxic nature of the peptides, it is possible and may be desirable to administer substantial excesses of these peptide compositions relative to these stated dosage amounts.

For treatment of chronic disease, a representative dose is in the range disclosed above, namely where the lower value is about 1, 5, 50, 500, or 1,000 μg of peptide and the higher value is about 10,000; 20,000; 30,000; or 50,000 μg of peptide, preferably from about 500 μg to about 50,000 μg of peptide per 70 kilogram patient. Initial doses followed by boosting doses at established intervals, e.g., from four weeks to six months, may be required, possibly for a prolonged period of time to effectively immunize an individual. In the case of chronic disease, administration should continue until at least clinical symptoms or laboratory tests indicate that the disease has been eliminated or substantially abated, and for a follow-up period thereafter. The dosages, routes of administration, and dose schedules are adjusted in accordance with methodologies known in the art.

The pharmaceutical, compositions for therapeutic treatment are intended for parenteral, topical, oral, intrathecal, or local administration. Preferably, the pharmaceutical compositions are administered parentally, e.g., intravenously, subcutaneously, intradermally, or intramuscularly.

Thus, in a preferred embodiment the invention provides compositions for parenteral administration which comprise a solution of the immunogenic peptides dissolved or suspended in an acceptable carrier, preferably an aqueous carrier. A variety of aqueous carriers may be used, e.g. water, buffered water, 0.8% saline, 0.3% glycine, hyaluronic acid and the like. These compositions may be sterilized by conventional, well known sterilization techniques, or may be sterile filtered. The resulting aqueous solutions may be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile solution prior to administration. The compositions may contain pharmaceutically acceptable auxiliary substances or pharmaceutical excipients as may be required to approximate physiological conditions, such as pH-adjusting and buffering agents, tonicity adjusting agents, wetting agents, preservatives, and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc.

The concentration of peptides of the invention in the pharmaceutical formulations can vary widely, i.e., from less than about 0.1%, usually at or at least about 2% to as much as 20% to 50% or more by weight, and will be selected primarily by fluid volumes, viscosities, etc., in accordance with the particular mode of administration selected.

A human unit dose form of the peptide composition is typically included in a pharmaceutical composition that also comprises a human unit dose of an acceptable carrier, preferably an aqueous carrier, and is administered in a volume of fluid that is known by those of skill in the art to be used for administration of such compositions to humans (see, e.g., Remington's Pharmaceutical Sciences, 17′ Edition, A. Gennaro, Editor, Mack Publishing Co., Easton, Pa., 1985).

The peptides of the invention can also be administered via liposomes, which serve to target the peptides to a particular tissue, such as lymphoid tissue, or to target selectively to infected cells, as well as to increase the half-life of the peptide composition. Liposomes include emulsions, foams, miceles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers and the like. In these preparations, the peptide to be delivered is incorporated as part of a liposome, alone or in conjunction with a molecule which binds to a receptor prevalent among lymphoid cells (such as monoclonal antibodies which bind to the CD45 antigen) or with other therapeutic or immunogenic compositions. Thus, liposomes either filled or decorated with a desired peptide of the invention can be directed to the site of lymphoid cells, where the liposomes then deliver the peptide compositions. Liposomes for use in accordance with the invention are formed from standard vesicle-forming lipids, which generally include neutral and negatively charged phospholipids and a sterol, such as cholesterol. The selection of lipids is generally guided by consideration of, e.g., liposome size, acid lability and stability of the liposomes in the blood stream. A variety of methods are available for preparing liposomes, as described in, e.g., Szoka, et al., Ann. Rev. Biophys. Bioenig. 9:467 (1980), and U.S. Pat. Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369.

For targeting compositions of the invention to cells of the immune system, a ligand can be incorporated into the liposome, e.g., antibodies or fragments thereof specific for cell surface determinants of the desired immune system cells. A liposome suspension containing a peptide may be administered intravenously, locally, topically, etc. in a dose which varies according to, inter alia, the manner of administration, the peptide being delivered, and the stage of the disease being treated.

For solid compositions, conventional nontoxic solid carriers may be used which include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like. For oral administration, a pharmaceutically acceptable nontoxic composition is formed by incorporating any of the normally employed excipients, such as those carriers previously listed, and generally 10-95% of active ingredient, that is, one or more peptides of the invention, often at a concentration of 25%-75%.

For aerosol administration, the immunogenic peptides are preferably supplied in finely divided form, along with a surfactant and propellant. Typical percentages of peptides are 0.011%-20% by weight, often 1%-10%. The surfactant must, of course, be pharmaceutically acceptable, and preferably soluble in the propellant. Representative of such agents are the esters or partial esters of fatty acids containing from 6 to 22 carbon atoms, such as caproic, octanoic, lauric, palmitic, stearic, linoleic, linolenic, olesteric and oleic acids with an aliphatic polyhydric alcohol or its cyclic anhydride. Mixed esters, such as mixed or natural glycerides may be employed. The surfactant may constitute 0.1%-20% by weight of the composition, preferably 0.25-5%. The balance of the composition is ordinarily propellant, although an atomizer may be used in which no propellant is necessary and other percentages are adjusted accordingly. A carrier can also be included, e.g., lecithin for intranasal delivery.

Antigenic peptides of the invention have been used to elicit a CTL and/or HTL response ex vivo, as well. The resulting CTLs or HTLs can be used to treat chronic infections, or tumors in patients that do not respond to other conventional forms of therapy, or who do not respond to a therapeutic peptide or nucleic acid vaccine in accordance with the invention. Ex vivo CTL or HTL responses to a particular antigen (infectious or tumor-associated) are induced by incubating in tissue culture the patient's, or genetically compatible, CTL or HTL precursor cells together with a source of antigen-presenting cells (APC), such as dendritic cells, and the appropriate immunogenic peptide. After an appropriate incubation time (typically about 7-28 days), in which the precursor cells are activated and expanded into effector cells, the cells are infused back into the patient, where they will destroy (CTL) or facilitate destruction (HTL) of their specific target cell (an infected cell or a tumor cell).

Kits

The peptide and nucleic acid compositions of this invention can be provided in kit form together with instructions for vaccine administration. Typically the kit would include desired composition(s) of the invention in a container, preferably in unit dosage form and instructions for administration. For example, a kit would include an APC, such as a dendritic cell, previously exposed to and now presenting peptides of the invention in a container, preferably in unit dosage form together with instructions for administration. An alternative kit would include a minigene construct with desired nucleic acids of the invention in a container, preferably in unit dosage form together with instructions for administration. Lymphokines such as IL-2 or IL-12 may also be included in the kit. Other kit components that may also be desirable include, for example, a sterile syringe, booster dosages, and other desired excipients.

The invention will be described in greater detail by way of specific examples. The following examples are offered for illustrative purposes, and are not intended to limit the invention in any manner. Those of skill in the art will readily recognize a variety of non-critical parameters that can be changed or modified to yield alternative embodiments in accordance with the invention.

EXAMPLES Example 1 Selection of Tumor Associated Antigens

Vaccines which bind to HLA supertypes, A2, A3, and B7, will afford broad, non-ethnically biased population coverage (83-88%). Vaccines which bind to HLA supertypes, A1, A11, and B44, will afford broad, non-ethnically biased population coverage (99-100%). Since the A2 supertype is broadly expressed in the population (39-49%), peptides which bind to this family of molecules provide a reasonable starting point for the use of peptide-based vaccines. While the A2 vaccine targets patients that express HLA-A2 molecules, the approach can be readily extended to include peptide(s) that bind to additional alleles or supertype groups thereof.

Whole proteins often induce an immune response limited to specific epitopes that may be ineffective in mediating effective anti-tumor immune responses (Disis et al., J. Immunology 156:3151-3158 (1996); Manca et al., J. Immunology 146:1964-1971 (1991)). An epitope-based vaccine circumvents this limitation through the identification of peptide epitopes embedded in TAAs. Exemplary TAAs are set forth in Table 12.

Peptides were evaluated based upon MHC binding motifs, on the capacity to bind MHC molecules, and the ability to activate tumor-reactive CTL in vitro using lymphocyte cultures from normal individuals. This approach has several advantages. First, it does not require the isolation of patient-derived cells such as CTL or tumor cells. Secondly, the identification of epitopes that stimulate CTL in normal individuals permits the identification of a broad range of epitopes, including subdominant as well as dominant epitopes.

Four tumor-associated antigens, CEA, p53, MAGE 2/3 and HER2/neu, are expressed in various tumor types (Kawashima et al., Human Immunology 59:1-14 (1998); Tomlinson, et al., Advanced Drug Delivery Reviews, Vol. 32(3) (6 Jul. 1998)). In a preferred embodiment, a vaccine comprises epitopes (as one or more peptides or as nucleic acids encoding them) from among these four, or any other, TAAs. Accordingly, this vaccine induces CTL responses against several major cancer types.

Carcinoembryonic antigen is a 180 kDmw cell surface and secreted glycoprotein overexpressed on most human adenocarcinomas. These include colon, rectal, pancreatic and gastric (Muraro, 1985) as well as 50% of breast (Steward, 1974) and 70% of non-small cell lung carcinomas (Vincent, 1978). This antigen is also expressed on normal epithelium and in some fetal tissue (Thompson, 1991).

The HER2/neu antigen (185 kDa) is a transmembrane glycoprotein with tyrosine kinase activity whose structure is similar to the epidermal growth factor receptor (Coussens, 1985; Bargmann, 1986; Yamamoto, 1986). Amplification of the HER2/neu gene and/or overexpression of the associated protein have been reported in many human adenocarcinomas of the breast (Slamon, 1987 and 1989; Borg, 1990), ovary (Slamon, 1989), uterus (Ber-chuck, 1991; Lukes, 1994), prostate (Kuhn, 1993; Sadasivan, 1993), stomach (Yonemura, 1991; Kameda, 1990; Houldsworth, 1990), esophagus (Houldsworth, 1990), pancreas (Yamanaka, 1993), kidney (Weidner, 1990) and lung (Kern, 1990; Rachwal, 1995).

The MAGE, melanoma antigen genes, are a family of related proteins that were first described in 1991. Van der Bruggen and co-workers were able to identify the MAGE gene after isolating CTLs from a patient who demonstrated spontaneous tumor regression. These CTLs recognized melanoma cell lines as well as tumor lines from other patients all expressing the same HLA-A1 restricted gene (van der Bruggen, 1991; De Plaen, 1994). The MAGE genes are expressed in metastatic melanomas (Brasseur, 1995), non-small lung (Weynants, 1994), gastric (Inoue, 1995), hepatocellular (Chen, 1999), renal (Yamanaka, 1998) colorectal (Mori, 1996), and esophageal (Quillien, 1997) carcinomas as wells as tumors of the head and neck (Lee, 1996), ovaries (Gillespie, 1998; Yamada, 1995), bladder (Chaux, 1998) and bone (Sudo, 1997). They are also expressed on normal tissue, specifically placenta and male germ cells (De Plaen, 1994). However, these normal cells do not express MHC Class I molecules and therefore do not present MAGE peptides on their surface.

In this study and previous work to identify A2 superfamily epitopes (Kawashima, 1998), MAGE-2 and MAGE-3 were considered a single TAA, based on the expression patterns and predicted primary amino acid sequences of the two genes. These two members of the MAGE family appear to be coordinately regulated (Zakut, 1993), resulting in a distribution in cancers that appears to be very similar, if not identical. Therefore, immune responses directed at either antigen should provide coverage for treatment of the cancers expected to express these TAA. The MAGE-2 and MAGE-3 proteins are 84% identical at the primary amino acid level. As a result, some epitopes are identical in the two antigens, while others are unique to one or the other. It should be noted that two subtypes of MAGE-2, designated “a” and b”, have been reported (Zakut, 1993). The gene referred to herein as MAGE-2 corresponds to the MAGE-2a subtype (C. Dahlberg personal communication, NB 1056, p. 16; Van der Bruggen, 1991; Zakut, 1993).

The fourth TAA selected for use in the vaccine is p53. In normal cells the p53 gene induces a cell cycle arrest which allows DNA to be checked for irregularities and maintains DNA integrity (Kuerbitz, 1992). Mutations in the gene abolish its suppressor function and allow escape of transformed cells from the restriction of controlled growth. At the same time, these mutations lead to overexpression of both wildtype and mutated p53 (Levine, 1991) making it more likely that epitopes within the protein may be recognized by the immune system. The most common mutations are at positions 175, 248, 273 and 282 and have been observed in colon (Rodrigues, 1990), lung (Fujino, 1995), prostate (Eastham, 1995), bladder (Vet, 1995) and bone cancers (Abudu, 1999; Hung, 1997).

Other TAAs that can be included in a vaccine composition are associated with prostate cancer (see, e.g., copending U.S. Patent Application U.S. Ser. No. 09/633,364, filed 8 Jul. 2000).

Table 7 below delineates the tumor antigen expression in breast, colon and lung. By targeting four TAA, the likelihood of the mutation of tumor cells (tumor escape) into cells which do not express any of the tumor antigens is decreased. Preferably, the inclusion of two or more epitopes from each TAA serves to increase the likelihood that individuals of different ethnicity will respond to the vaccine and provides broadened population coverage.

This rational approach to vaccine compositions can be focused on a particular HLA allele, or extended to various HLA molecules or supertypes to further extend population coverage.

Table 8 shows the incidence, 5-year survival rates, and the estimated number of deaths per year for these tumors in the U.S for each type of cancer in Table 7. In terms of estimated new cases, estimated deaths and 5 year survival rates each of these tumor types has a large unmet need. Globally, the incidence of these tumors is significantly greater.

Example 2 Identification of A2 Supermotif/Motif-Bearing Peptides

Protein sequences from the four targeted tumor antigens (CEA, p53, MAGE 2/3 and HER2/neu) were analyzed, to identify 8-, 9-, 10-, and 11-mer sequences containing the HLA-A2 supertype binding motif. This motif [leucine (L), isoleucine (I), valine (V), methionine (M), alanine (A), threonine (T), or glutamine (Q) at position 2, and leucine (L), isoleucine (I), valine (V), methionine (A, alanine (A), or threonine (T) at the C-terminus; see Table 2] is the predominant factor in determining peptide binding to the HLA molecules within the A2 supertype (see, e.g., del Guercio et al., J. Immunol., 154:685-693 (1995); Sette, A. and Sidney, J., Cur. Opin. Immunol., 10: 478-482 (1998); Sidney et al., Immunology Today, 17:261-266 (1996)). Nonamer and decamer sequences were further characterized using an A2-specific algorithm to evaluate secondary anchor residues (Ruppert et al., Cell 74:929-937 (1993); Gulukota et al., J. Mol. Biol. 267:1258-1267 (1997)).

Example 3 Molecular Binding Assays

Native sequences containing HLA-A2 peptide motifs were tested directly for binding to human class I HLA molecules, since a subset of motif-bearing peptides bind with a biologically significant affinity, data depicted in Table 6. An affinity threshold ≦500 nM to the HLA-A2 molecule was previously shown to define the capacity of a peptide epitope to elicit a CTL response (Sette et al., J. Immunol. 153:5586-5592 (1994)). A competitive inhibition assay using purified HLA molecules was used to quantify peptide binding. Motif-bearing peptides were initially tested for binding to HLA-A*0201, the prototype member of the HLA-A2 supertype. Peptides binding to A*0201 with an IC₅₀≦500 nM were subsequently tested for their capacity to bind other predominant molecules of the A2 supertype: A*0202, A*0203, A*0206 and A*6802 (del Guercio et al., J. Immunol., 154:685-693 (1995); Sette, A. and Sidney, J., Cur. Opin. Immunol., 10: 478-482 (1998); Sidney et al., Immunology Today, 17:261-266 (1996)). A*0201-binding peptides found to bind at least one additional A2 supertype member were selected for further testing. Analogs of the native sequences for the CEA and p53 were evaluated to identify additional CTL peptide epitopes, as described below.

Example 4 A2 Epitope Identification

Since HLA-A2 is a species restricted molecule, the binding and functional activities of the A2 vaccine epitopes were measured in vitro using human molecules and cells. CTL epitopes were identified that demonstrated high or intermediate HLA-A2 binding affinity (IC₅₀ of ≦500 nM). These epitopes also bound to at least one additional member of the HLA-A2 supertype family with an IC₅₀≦500 nM. Each epitope stimulated the in vitro induction of a specific human CTL that recognized and lysed peptide-pulsed target cells and tumor cell lines expressing the relevant TAA. A PADRES molecule is optionally included in the vaccine to promote the induction of long lasting CTL responses (Alexander et al., Immunol. Res. 18(2):79-92 (1998)).

Immunological responses were demonstrated by in vitro induction of human CTL that were capable of recognizing both peptide-pulsed cells and TAA-expressing tumor cell lines. In certain cases, analog peptides were selected based on either improved binding affinity or supertype coverage relative to the native peptide and in one case, substitution of a cysteine with another amino acid.

Analogous assays can be used for other HLA types.

Example 5 Peptide Analogs Increase Supertype Cross-Reactivity or Improve Chemical Characteristics

Class I HLA peptides can be modified, or “analoged” by substitution of amino acids at a given position to increase their HLA binding affinity and/or supertype cross-reactivity (see, e.g., Table 2, and Zitvogel et al., J Exp Med 183:87-97 (1996); Sette, et al., J. Immunol. 153:5586-5592 (1994)). The amino acids at position 2 and the C terminus of a peptide are the primary contact or “anchor” residues that interact with the HLA-A2 binding pocket. In order to identify analogs for inclusion in a composition of the invention, anchor residues were modified by substitution with a presently preferred or less preferred anchor residue, at position 2 and/or at the C-terminus.

Another type of modification utilized involved the substitution of α-amino butyric acid (B) for endogenous cysteine (C) residues to avoid the potential complication of disulfide bridge formation during experimentation and development.

For example, two criteria that were used to select native peptides to be analoged: 1) presence of a suboptimal anchor residue; and 2) at least weak binding (IC₅₀=500-5000 nM) of the parent peptide to at least two or three alleles of a supertype.

Peptides can also be analoged by modification of a secondary anchor residue. For example, in preferred approaches, a peptide can be analoged by removal of a deleterious residue in favor of an acceptable or preferred one; an acceptable residue can be exchanged for a different acceptable residue or a preferred residue, or a preferred residue can be exchanged for another preferred one.

Accordingly, peptide sequences were modified using one or more of the strategies described above. The peptides were tested for HLA-A2 supertype binding using the molecular binding assay. Supertype-binding data for analog peptides are shown in Table 6.

Example 6 Cellular Immunogenicity Screening

The peptides of the invention were also evaluated for their potential to stimulate CTL precursor responses to the TAA-derived peptide (in vitro primary CTL induction) and CTL recognition of tumor cells expressing the target TAA peptide epitope (recognition of endogenous targets). These criteria provided evidence that the peptides are functional epitopes.

In Vitro Primary CTL Induction

Peripheral blood monocytic cell-derived (or bone-marrow-derived) human DC, generated in vitro using GM-CSF and IL-4 and pulsed with a peptide of interest, were used as antigen presenting cells (APCs) in primary CTL induction cultures. The peptide pulsed DC were incubated with CD8 T cells (positively selected from normal donor lymphocytes using magnetic beads) which served as the source of CTL precursors. One week after stimulation with peptide, primary cultures were tested for epitope-specific CTL activity using either a standard chromium-release assay which measures cytotoxicity or a sandwich ELISA-based interferon gamma (IFNγ) production assay. Each of the CTL epitopes of Table 6 stimulated CTL induction from CD8 T cells of normal donors.

Recognition of Endogenous Targets

As described herein, T cell cultures testing positive for recognition of peptide-pulsed targets were expanded and evaluated for their ability to recognize human tumor cells that endogenously express the TAA. The chromium-release and IFNγ production assays were used for these evaluations, with tumor cell lines serving as the targets. Tumor cell lines lacking expression of either the TAA or the HLA-A2.1 molecule served as the negative control for non-specific activity. CTL cultures were generated which recognized tumor cells in a peptide-specific and HLA-A2-restricted manner (Table 6).

The HLA receptor binding and immunogenicity characteristics of CTL peptides are summarized in Table 6.

Example 7 A PADRE® Molecule as a Helper Epitope for Enhancement of CTL Induction

There is increasing evidence that HTL activity is critical for the induction of long lasting CTL responses (Livingston et al. J. Immunol 162:3088-3095 (1999); Walter et al., New Engl. J. Med. 333:1038-1044 (1995); Hu et al., J. Exp. Med. 177:1681-1690 (1993)). Therefore, one or more peptides that bind to HLA class II molecules and stimulate HTLs can be used in accordance with the invention. Accordingly, a preferred embodiment of a vaccine includes a molecule from the PADRE® family of universal T helper cell epitopes (HTL) that target most DR molecules in a manner designed to stimulate helper T cells. For instance, a pan-DR-binding epitope peptide having the formula: aKXVAAZTLKAAa, where “X” is either cyclohexylalanine, phenylalanine, or tyrosine; “Z” is either tryptophan, tyrosine, histidine or asparagine; and “a” is either D-alanine or L-alanine (SEQ ID NO:29), has been found to bind to most HLA-DR alleles, and to stimulate the response of T helper lymphocytes from most individuals, regardless of their HLA type.

A particularly preferred PADRE® molecule is a synthetic peptide, aKXVAAWTLKAAa (a=D-alanine, X=cyclohexylalanine), containing non-natural amino acids, specifically engineered to maximize both HLA-DR binding capacity and induction of T cell immune responses.

Alternative preferred PADRE® molecules are the peptides, aKFVAAWTLKAAa, aKYVAAWTLKAAa, aKFVAAYTLKAAa, aKXVAAYTLKAAa, aKYVAAYTLKAAa, aKFVAAHTLKAAa, aKXVAAHTLKAAa, aKYVAAHTLKAAa, aKFVAANTLKAAa, aKXVAANTLKAAa, aKYVAANTLKAAa, AKXVAAWTLKAAA (SEQ ID NO:30), AKFVAAWTLKAAA (SEQ ID NO:31), AKYVAAWTLKAAA (SEQ ID NO:32), AKFVAAYTLKAAA (SEQ ID NO:33), AKXVAAYTLKAAA (SEQ ID NO:34), AKYVAAYTLKAAA (SEQ ID NO:35), AKFVAAHTLKAAA (SEQ ID NO:36), AKXVAAHTLKAAA (SEQ ID NO:37), AKYVAAHTLKAAA (SEQ ID NO:38), AKFVAANTLKAAA (SEQ ID NO:39), AKXVAANTLKAAA (SEQ ID NO:40), AKYVAANTLKAAA (SEQ ID NO:41) (a=D-alanine, X=cyclohexylalanine).

In a presently preferred embodiment, the PADRE® peptide is amidated. For example, a particularly preferred amidated embodiment of a PADRE® molecule is conventionally written aKXVAAWTLKAAa-NH₂.

Competitive inhibition assays with purified HLA-DR molecules demonstrated that the PADRE® molecule aKXVAAWTLKAAa-NH₂ binds with high or intermediate affinity (IC₅₀≦1,000 nM) to 15 out of 16 of the most prevalent HLA-DR molecules ((Kawashima et al., Human Immunology 59:1-14 (1998); Alexander et al., Immunity 1:751-761 (1994)). A comparison of the DR binding capacity of PADRE® and tetanus toxoid (TT) peptide 830-843, a “universal” epitope has been published (Panina-Bordignon et al., Eur. J. Immunology 19:2237-2242 (1989)). The TT 830-843 peptide bound to only seven of 16 DR molecules tested, while PADRE® bound 15 of 16. At least 1 of the 15 DR molecules that bind PADRE® is predicted to be present in >95% of all humans. Therefore, this PADRE® molecule is anticipated to induce an HTL response in virtually all patients, despite the extensive polymorphism of HLA-DR molecules in the human population.

PADRE® has been specifically engineered for optimal immunogenicity for human T cells. Representative data from in vitro primary immunizations of normal human T cells with TT 830-843 antigen and the PADRE® molecule aKXVAAWTLKAAa-NH₂ are shown in FIG. 1. Peripheral blood mononuclear cells (PBMC) from three normal donors were stimulated with the peptides in vitro. Following the third round of stimulation, it was observed that PADRE® generated significant primary T cell responses for all three donors as measured in a standard T cell proliferation assay. With the PADRE® peptide, the 10,000 cpm proliferation level was generally reached with 10 to 100 ng/ml of antigen. In contrast, TT 830-843 antigen generated responses for only 2 out of 3 of the individuals tested. Responses approaching the 10,000 cpm range were reached with about 10,000 ng/ml of antigen. In this respect, it was noted that PADRE® was, on a molar basis, about 100-fold more potent than TT 830-843 antigen for activation of T cell responses.

Early data from a phase I/II investigator-sponsored trial, conducted at the University of Leiden (C. J. M. Melief), support the principle that the PADRE® molecule aKXVAAWTLKAAa, possibly the amidated aKXVAAWTLKAAa-NH₂, is highly immunogenic in humans (Ressing et al., Detection of immune responses to helper peptide, but not to viral CTL epitopes, following peptide vaccination of immunocompromised patients with recurrent cervical carcinoma. (J. Immunother. 23(2):255-66 (2000)). In this trial, a PADRE® molecule was co-emulsified with various human papilloma virus (HPV)-derived CTL epitopes and was injected into patients with recurrent or residual cervical carcinoma However, because of the late stage of carcinoma with the study patients, it was expected that these patients were immunocompromised. The patients' immunocompromised status was demonstrated by their low frequency of influenza virus-specific CTL, reduced levels of CD3 expression, and low incidence of proliferative recall responses after in vitro stimulation with conventional antigens. Thus, no efficacy was anticipated in the University of Leiden trial, rather the goal of that trial was essentially to evaluate safety. Safety was, in fact, demonstrated. In addition to a favorable safety profile, PADRE® T cell reactivity was detected in four of 12 patients (FIG. 2) in spite of the reduced immune competence of these patients.

Thus, the PADRE® peptide component(s) of the vaccine bind with broad specificity to multiple allelic forms of HLA-DR molecules. Moreover, PADRE® peptide component(s) bind with high affinity (IC₅₀≦1000 nM), i.e., at a level of affinity correlated with being immunogenic for HLA Class II restricted T cells. The in vivo administration of PADRE® peptide(s) stimulates the proliferation of HTL in normal humans as well as patient populations.

Example 8 Functional Competence of ProGP-Derived DC

One embodiment of a vaccine in accordance with the invention comprises epitope-bearing peptides of the invention delivered via dendritic cells (DC). Accordingly, DC were evaluated in both in vitro and in vivo immune function assays. These assays include the stimulation of CTL hybridomas and CTL cell lines, and the in vivo activation of CTL.

DC Purification

ProGP-mobilized DC were purified from peripheral blood (PB) and spleens of ProGP-treated C57B1/6 mice to evaluate their ability to present antigen and to elicit cellular immune responses. Briefly, DC were purified from total WBC and spleen using a positive selection strategy employing magnetic beads coated with a CD11c specific antibody (Miltenyi Biotec, Auburn Calif.). For comparison, ex vivo expanded DC were generated by culturing bone marrow cells from untreated C57B1/6 mice with the standard cocktail of GM-CSF and IL-4 (R&D Systems, Minneapolis, N) for a period of 7-8 days (Mayordomo et al., Nature Med. 1:1297-1302 (1995)). Recent studies have revealed that this ex vivo expanded DC population contains effective antigen presenting cells, with the capacity to stimulate anti-tumor immune responses (Celluzzi et al., J. Exp. Med. 83:283-287 (1996)).

The purities of ProGP-derived DC (100 μg/day, 10 days, SC) and GM-CSF/IL-4 ex vivo expanded DC were determined by flow cytometry. DC populations were defined as cells expressing both CD11c and MHC Class II molecules. Following purification of DC from magnetic CD11c microbeads, the percentage of double positive PB-derived DC, isolated from ProGP-treated mice, was enriched from approximately 4% to a range from 48-57% (average yield=4.5×10⁶ DC/animal). The percentage of purified splenic DC isolated from ProGP treated mice was enriched from a range of 12-17% to a range of 67-77%. The purity of GM-CSF/IL-4 ex vivo expanded DC ranged from 31-41% (Wong et al., J. Immunother., 21:32040 (1998)).

In Vitro Stimulation of CTL Hybridomas and CTL Cell Lines: Presentation of Specific CTL Epitopes

The ability of ProGP generated DC to stimulate a CTL cell line was demonstrated in vitro using a viral-derived epitope and a corresponding epitope responsive CTL cell line. Transgenic mice expressing human HLA-A2.1 were treated with ProGP. Splenic DC isolated from these mice were pulsed with a peptide epitope derived from hepatitis B virus (HBV Pol 455) and then incubated with a CTL cell line that responds to the HBV Pol 455 epitope/HLA-A2.1 complex by producing IFNγ. The capacity of ProGP-derived splenic DC to present the HBV Pol 455 epitope was greater than that of two positive control populations: GM-CSF and IL-4 expanded DC cultures, or purified splenic B cells (FIG. 3). The left shift in the response curve for ProGP-derived spleen cells versus the other antigen presenting cells reveal that these ProGP-derived cells require less epitope to stimulate maximal IFNγ release by the responder cell line.

Example 9 Peptide-Pulsed ProGP-Derived DC Promote in Vivo CTL Responses

The ability of ex vivo peptide-pulsed DC to stimulate CTL responses in vivo was also evaluated using the HLA-A2.1 transgenic mouse model. DC derived from ProGP-treated animals or control DC derived from bone marrow cells after expansion with GM-CSF and IL-4 were pulsed ex vivo with the HBV Pol 455 CTL epitope, washed and injected (IV) into such mice. At seven days post immunization, spleens were removed and splenocytes containing DC and CTL were restimulated twice in vitro in the presence of the HBV Pol 455 peptide. The CTL activity of three independent cultures of restimulated spleen cell cultures was assessed by measuring the ability of the CTL to lyse ⁵¹Cr-labeled target cells pulsed with or without peptide. Vigorous CTL responses were generated in animals immunized with the epitope-pulsed ProGP derived DC as well as epitope-pulsed GM-CSF/IL-4 DC (FIG. 4). In contrast, animals that were immunized with mock-pulsed ProGP-generated DC (no peptide) exhibited no evidence of CTL induction. These data confirm that DC derived from ProGP treated mice can be pulsed ex vivo with epitope and used to induce specific CTL responses in vivo. Thus, these data support the principle that ProGP-derived DC promote CTL responses in a model that manifests human MHC Class I molecules.

In vivo pharmacology studies in mice have demonstrated no apparent toxicity of reinfusion of pulsed autologous DC into animals.

Example 10 Manufacturing of Synthetic Peptides

Physical/Chemical Properties of the Bulk A2 Vaccine Peptides

In one embodiment, each peptide of the invention is prepared by chemical synthesis and is isolated as a solid by lyophilization. Peptides are manufactured in compliance with Good Manufacturing Practices.

Bulk peptides of the invention, following identity and release testing, are formulated as an aqueous or non-aqueous solution, sterile filtered, and aseptically filled into sterile, depyrogenated vials. Sterile rubber stoppers are inserted and overseals applied to the vials. The vialed formulations undergo 100% visual inspection and specified release testing. The released vials are labeled and packaged before delivery for administration.

Table 6 summarizes the identifying source number, the amino acid sequence, binding data, and properties of CTLs induced by each peptide.

Example 11 Dendritic cell Isolation, Pulsing, Testing and Administration

A presently preferred procedure for vaccination is set forth herein. In brief, patients are treated with ProGP to expand and mobilize DC into the circulation. On the day of peak DC mobilization, determined in accordance with procedures known in the art, patients undergo leukapheresis (approximately 15 L process, possibly repeated once if required to collect sufficient mononuclear cells). The mononuclear cell product is admixed with peptides of the invention by injection through micropore filters (this admixing protocol is not needed if sterile peptides are used). After incubation and washing to remove residual unbound peptides, the cell product vaccine embodiment is resuspended in cryopreservative solution (final 10% DMSO) and, for those protocols involving multiple vaccination boosts, divided into aliquots. The pulsed mononuclear cell product(s) are frozen and stored according to accepted procedures for hematopoietic stem cells.

Vaccination is performed by injection or intravenous infusion of thawed cell product after the hematologic effects of ProGP in the patient have dissipated (i.e., the hemogram has returned to baseline). FIG. 5 provides a flow chart of ex vivo pulsing of DC with peptides, washing of DC, DC testing, and cryopreservation. A more detailed description of the process is provided in the following Examples.

Example 12 Administration of ProGP and Collection of Mononuclear Cells by Leukapheresis

Patients are treated with ProGP daily by subcutaneous injection (dose and schedule determined in accordance with standard medical procedures). On the evening before leukapheresis, patients are assessed by an apheresis physician or nurse/technologist for adequacy of intravenous access for large-bore apheresis catheters. If peripheral venous access is deemed inadequate to maintain rapid blood flow for apheresis, then central venous catheters (inguinal, subclavian or internal jugular sites) can be inserted by appropriate medical/surgical personnel. On the day of predicted peak DC mobilization, leukapheresis (approximately 3 blood volumes or 15 L) is performed, for example, on a Cobe Spectra or Fenwal CS3000 (flow rate ≧35 mL/min) to obtain mononuclear cells. The number of DC in the leukapheresis product is estimated by flow cytometric counting of mononuclear cells possessing the immunophenotypes lin-/HLA-DR+/CD11c+ and lin-/HLA-DR+/CD123+ in a 1 mL sample aseptically withdrawn from the apheresis product. The numbers of granulocytes and lymphocytes in the leukapheresis product are counted by automated cytometry (CBC/differential). CBC/differential is performed immediately after the leukapheresis procedure and every other day for ten days to monitor resolution of the hematologic effects of the hematopoietin treatment and apheresis.

Example 13 A Procedure for Dendritic Cell Pulsing

Plasma is removed from the leukapheresis product by centrifugation and expression of supernatant. The cells from the centrifugation pellet are resuspended in OptiMEM medium with 1% Human Serum Albumin (HSA) at a cell density of 10⁷ DC/ml in up to 100 ml.

The peptide(s) of the invention, preferably as individual sterile A2 peptide formulations, are administered directly into the DC culture bag through an injection port, using aseptic technique. After mixing, e.g., by repeated squeezing and inversion, the cell suspension is incubated for four hours at ambient temperature. Cryopreservative solution is prepared by dissolving 50 mL pharmaceutical grade dimethylsulfoxide (DMSO) in 200 mL Plasmalyte®. After the pulsing period, the cell suspension is washed by centrifugation and resuspension in an equal volume of phosphate buffered saline solution. The washing procedure is repeated a defined number of times, e.g., until studies validate that peptides have been removed. Samples of one milliliter each are removed for viability testing and microbiological testing. The cells are then prepared for freezing by centrifugation and resuspension in an equal volume of cryopreservative solution (final 10% DMSO). The cell suspension in cryopreservative is then divided into six equal aliquots, transferred to 50 ml freezing bags (Fenwal) and frozen at controlled rate of 1° C./min for storage in liquid nitrogen until needed for vaccination procedure.

Assay to Evaluate the Pulsing Procedure

Antigen presenting cells, long-term stimulated T cells corresponding to peptides of the invention, or T cell hybridomas, are used to determine the optimal procedure for incubating the peptide reagents of a vaccine with human cells. Pulsing studies are done using one or more of the following cell sources: purified DC from ProGP treated HLA-A2.1 transgenic mice; human tumor cell lines that express HLA-A2; peripheral blood mononuclear cells from normal human volunteers; peripheral blood mononuclear cells from ProGP treated patients; and/or DC obtained from normal human HLA-A2 volunteers following the ex vivo culture of their peripheral blood mononuclear cells with GM-CSF and IL-4.

Evaluated conditions include, e.g.:

-   -   Cellular isolation procedure and cell number     -   Concentration of vaccine peptides     -   Washing conditions to remove ancillary reagents     -   Post-pulsing manipulations (resuspension, freezing)

Accordingly, these studies demonstrate the ability of the procedure to produce functional HLA-A2/peptide complexes on the surface of the human cells. The validation of the pulsing procedure is established using HLA-A2.1-specific T cell lines after which the Phase I clinical trial occurs.

This Example may also be performed using A1, A3, A24, B7 or B44-restricted peptides by substituting appropriate HLA-related reagents. It will be clear to one of skill in the art how to make such substitutions.

Example 14 Validation of Peptide Removal from the DC Product

Following pulsing with the peptide reagents, DC from the patient are washed several times to remove excess peptides prior to infusing the cells back into the patient. In this embodiment of a vaccine of the invention, the washing procedure removes unbound peptides. Accordingly, there is no, or negligible, systemic exposure of the patient to the peptides. Alternative vaccines of the invention involve direct administration of peptides of the invention to a patient, administration of a multiepitopic polypeptide comprising one or more peptides of the invention, administration of the peptides in a form of nucleic acids which encode them, e.g., by use of minigene constructs, or by viral vectors.

Assay for Vaccine Peptides in the Dendritic Cell Wash Buffer

After the DC are incubated with the peptides, the cells are washed with multiple volumes of wash buffer. An aliquot of the last wash is placed onto a nonpolar solid-phase extraction cartridge and washed to reduce the salt content of the sample. Any peptides contained in the buffer will be eluted from the extraction cartridge and evaporated to dryness. The sample is then reconstituted in High Performance Liquid Chromatography (HPLC) mobile phase, injected onto a polymer based reverse-phase HPLC column, and eluted using reverse-phase gradient elution chromatography. Residual peptides are detected using a mass spectrometer set-up to monitor the protonated molecular ions of each peptide as they elute from the HPLC column. The peptides are quantified by comparing the area response ratio of analyte and internal standard to that obtained for standards in a calibration curve.

Example 15 Validation of Trifluoroacetic Acid Removal from the DC Product

In a particular embodiment, peptide reagents may be formulated using 0.1% trifluoroacetic acid (TFA). The washing procedure developed to remove residual peptide also removes residual TFA.

Example 16 Dendritic Cell Release Testing

Identity

The number of DC in the leukapheresis product is estimated by flow cytometric counting of mononuclear cells possessing the immunophenotypes lin⁻/HLA-DR⁺/CD11c⁺ and lin⁻/HLA-DR⁺/CD123⁺ in a 1 ml sample aseptically withdrawn from the apheresis product. Lin⁻ cells excludes monocytes, T-lymphocytes, B-lymphocytes, and granulocytes, by using a cocktail of antibodies to lineage markers CD3, CD14, DC16, CD19, CD20, CD56.

Cell Viability

Viability of mononuclear cells is assessed after pulsing and washing, prior to suspension in cryopreservative, by trypan blue dye exclusion. In general, if the cell product contains more than 50% trypan blue-positive cells, the product is not administered to a patient.

Microbiological Testing

The cell suspension in cryopreservative is examined for microbial contamination by gram stain and routine clinical bacterial and fungal culture/sensitivity. If tests are positive for bacterial or fungal contamination, implicit evidence of significant contamination, the product is not infused. If, e.g., a gram stain is negative, the product may be infused for the first vaccination while awaiting results of culture/sensitivity. Antibiotic therapy based on culture results is instituted at the discretion of the treating physician if the patient shows appropriate signs of infection that could be clinically attributable to the infused contaminant.

Example 17 Patient Vaccination

In a preferred embodiment, an aliquot of frozen pulsed dendritic cell product is removed from a liquid nitrogen freezer and kept frozen in an insulated vessel containing liquid nitrogen during transport to the infusion site. The product is thawed by immersion with gentle agitation in a water bath at 37° C. Immediately on thawing, the cell suspension is infused through intravenous line by gravity or by syringe pump. Alternatively, the vaccine is administered by injection, e.g., subcutaneously, intradermally, or intramuscularly. The patient's vital signs are monitored before infusion/injection and at 5 minute intervals during an infusion, then at 15 minute intervals for 1 hour after infusion/injection.

Infusion protocols in accordance with knowledge in the art are carried out for alternative vaccine embodiments of the invention, such as direct peptide infusion or nucleic acid administration.

Example 18 An A2 Vaccine

A vaccine in accordance with the invention comprises eight peptide epitopes bearing the HLA-A2 supermotif. Collectively, these eight epitopes are derived from the tumor associated antigens (TAAs) HER2/neu, p53, MAGE 2, MAGE3, and carcinoembryonic antigen (CEA), and stimulate CTL responses to these TAAs. (see Table 9) These eight peptides, which are also presented in Table 6, bear an HLA-A2 supermotif. Optionally, a ninth peptide, an HTL epitope that enhances CTL responses such as a pan-DR-binding peptide (PADRE®, Epimmune, San Diego, Calif.), is included.

The eight HLA-A2 peptide components of the A2 vaccine bind to multiple HLA-A2 superfamily molecules with high or intermediate affinity (IC₅₀≦500 nM). HLA-A2-specific analog and native peptide components of the A2 vaccine stimulate CTL from the peripheral blood of normal human volunteers. These CTL recognize native peptides that have been pulsed onto HLA-A2 expressing APCs, as well as endogenous peptides presented by HLA-matched tumor cell lines. Thus, the A2 vaccine is effective in stimulating the cellular arm of the immune system to mediate immune responses against tumors.

It is to be appreciated that vaccines comprising peptides bearing other motifs, or nucleic acids encoding such peptides, are also used in accordance with the principles set forth herein, and are within the scope of the present invention.

In a preferred embodiment, an A2 vaccine comprises DC pulsed ex vivo with the nine peptides. This embodiment of a vaccine can be used with progenipoietin (ProGP)-mobilized DC.

Example 19 An A2 Vaccine

An A2 vaccine comprises a cocktail of 12 peptides, 10 of which stimulate CTL responses to the tumor associated antigens (TAA) HER2/neu, p53, MAGE 2/3, and carcinoembryonic antigen (CEA). The remaining two peptides are both members of the PADRE® family of peptides that are HTL epitopes that enhance CTL responses (see Table 10). This embodiment of an A2 Vaccine is used in combination with an emulsion-based adjuvant such as Montanide® ISA51 or ISA720 (Seppic, Paris, France) or an Incomplete Freund's Adjuvant, preferably administered by injection. As appreciated by those of skill in the art, alternative modes of administration can also be used. Many adjuvants are known in the art, and are used in accordance with the present invention, see, e.g., Tomlinson, et al., Advanced Drug Delivery Reviews, Vol. 32(3) (6 Jul. 1998).

The eight HLA-A2 CTL, peptide components of this vaccine embodiment bind to multiple HLA-A2 superfamily molecules with high or intermediate affinity (IC₅₀≦500 nM). The HLA-A2-specific analog and native peptide components of the present vaccine stimulate CTL from patient's blood. These CTL recognize native peptides that were pulsed onto HLA-A2 expressing APCs, as well as endogenous peptides presented by HLA-matched tumor cell lines.

Two peptides that stimulate HLA class II are also used in accordance with the invention. For instance, a pan-DR-binding epitope peptide having the formula: aKXVAAZTLKAAa, where “X” is either cyclohexylalanine, phenylalanine, or tyrosine; “Z” is either tryptophan, tyrosine, histidine or asparagine; and “a” is either D-alanine or L-alanine (SEQ ID NO:29), has been found to bind to most HLA-DR alleles, and to stimulate the response of T helper lymphocytes from most individuals, regardless of their HLA type. Two particularly preferred PADRE® molecules are the peptides, aKFVAAYTLKAAa-NH₂ and aKXVAAHTLKAAa-NH₂ (a=D-alanine, X=cyclohexylalanine), the latter containing a non-natural amino acid, specifically engineered to maximize both HLA-DR binding capacity and induction of T cell immune responses.

The PADRE® peptide components of the A2 vaccine bind with high affinity and broad specificity to multiple allelic forms of HLA-DR molecules (IC₅₀≦1000 nM). The in vivo administration of PADRE® peptide stimulates the proliferation of HTL in normal humans as well as patient populations. Thus, this vaccine embodiment is effective in stimulating the cellular arm of the immune system to mediate immune responses against tumors.

Example 20 Identification of HLA-A1, -A3, -A24 and -B7 Motif Bearing Peptides

Cytotoxic T cells (CTLs) play a major role in anti-tumor immune responses by directly lysing tumor cells and also by secreting cytokines such as interleukin-2, TNFα (tumor necrosis factor), GM-CSF (granulocyte-macrophage colony stimulating factor) and interferon gamma (IFNγ) which can contribute to the anti-tumor effect. These CTLs recognize small peptides, 8-11 amino acids long, that are derived from antigens expressed specifically by tumor cells and bound to MHC Class I molecules (Zinkernagel, 1997; York, 1996; Rammensee, 1993). The role of CTLs in tumor regression has been documented in both mouse models and patients. The CTLs constitute a major component of immune lymphocytes infiltrating tumor sites (TIL cells). These cells have been associated with spontaneous tumor regression in humans (Zorn, 1999). In vivo CTL induction in transgenic mice gives rise to CTLs that recognize tumor cells resulting in tumor regression. Toes et al (1996) and Vierboom et al (1997) also performed adoptive transfer experiments in mice and observed protection from tumor development. Adoptive transfer experiments in humans have also demonstrated the efficacy of anti-tumor CTL (Greenberg, 1991; Kawakami, 1995). Human trials have demonstrated that epitope-specific CTLs can be induced in cancer patients and in several instances correlated their induction with partial or complete tumor responses (Murphy, 1996; Nestle, 1998; Rosenberg, 1998).

An important first step in the development of a cancer vaccine is the identification of these peptide epitopes from tumor-associated antigens (TAA). There are numerous tumor-associated antigens expressed by tumor cells and a tumor cell may express multiple epitopes from several TAA (Tsang, 1995; Rongcun, 1999; Soussi, 1996). Several investigators (Shu, 1997; Wang, 1997; Gilboa, 1999 and Berlyn, 1999) have attempted to categorize the tumor antigens identified to date. Briefly, these are differentiation antigens that correspond to normal tissue-specific gene products such as tyrosinase, gp100 and MART-1; mutations of tumor suppressor genes such as p53, ras and bcr/abl; overexpressed normal or embryonic gene products represented by MAGE (a family of melanoma associated antigens), Her2/neu and CEA; and antigens derived from viruses such as human papilloma virus. Tumor cells may be expressed by more than one TAA, and a tumor cell may express multiple epitopes from a particular TAA (Van den Eynde, 1989; Tsang, 1995; Rongcun, 1999; Soussi, 1996).

For the purpose of developing a broadly effective cancer vaccine, four TAA (CEA, HER2/neu, MAGE2/3, and p53) expressed by many tumors such as colon, breast, lung and gastric cancers, and in the case of MAGE, some melanomas, were selected. The use of multiple TAA should address the potential problem in developing a cancer immunotherapeutic; namely, that tumor escape can occur through the selection of antigen-negative variants (Boon, 1989a,b; Melief, 1989).

Using peptide epitopes in a vaccine composition has distinct advantages over using whole antigen. The whole antigen may include immunosuppressive epitopes or might have undesired intrinsic biological activity. An additional advantage to an epitope-based vaccine is the ability to combine both CTL and helper epitopes, or epitopes from multiple TAA or HLA types into a single formulation.

One obstacle in the development of CTL epitope based vaccines is the large degree of MHC polymorphism (Sette, 1998). The Class I MHC displaying these epitopes in humans are termed human leukocyte antigens, or HLA. While there are over 125 HLA Class I molecules, it has been discovered that most can be grouped into one of several families or “supertypes” based on their ability to bind similar repertoires of peptides. Nine major class I supertypes have been described (Sette and Sidney, 1999). Of these, the five most prevalent are HLA-A2.1, -A3, -B7, -A1 and -A24. Together these 5 supertypes cover, on average, 98.8% of the Caucasian, African American, Japanese, Chinese and Hispanic populations (Table 1a). The A2 superfamily and the corresponding peptide motifs have been characterized elsewhere (Sette, 1998). Therefore, this work will focus on HLA-A3, -B7, -A1 and -A24. The A3 superfamily comprises A*03, A*11, A*3101, A*3301 and A*6801, of which A*03 and A*11 are the most predominant. The A3 supertype provides an average coverage of 44.2% amongst the 5 major ethnicities: Caucasian, Black, Japanese, Chinese and Hispanic populations (Table 11, Sidney, 1996a). A B7 superfamily has also been identified and comprises HLA-B*0702, B*3501-3, B*51, B*5301 and B*5401, and has an average population coverage of 44.7% (Table 11, Sidney, 1996b). Work done at Epimmune and by others has demonstrated that many peptides exhibit degenerate (crossreactive) binding (Tanigaki, 1994; del Guercio, 1995; Sidney, 1995; Sidney, 1996b) which would allow us to identify supertype cross-reactive epitopes that would extend the breadth of coverage.

Until binding assays for all of the HLA-A1 and -A24 superfamily alleles are developed, identification of HLA-A1 and -A24 restricted candidate peptides relies on motif analysis and binding affinity assays for the primary alleles, A*0101 and A*2402. These 2 alleles would provide average population coverage of 11.9% and 28.7%, respectively (Table 11). HLA-A*01 increases coverage of black, Chinese, and Hispanic populations and A*24 provides significant coverage of the Asian and Hispanic populations.

Analysis of HCV-derived peptides revealed that peptides binding the predominant allele of the supertype (i.e. A*0201 for the A2 supertype and A*0301 for the A3 supertype) with an IC₅₀≦100 nM showed cross-reactive binding and were recognized by infected patients. Hepatitis B virus-derived peptides that fit the same criteria were also demonstrated to be immunogenic 89% of the time either in transgenic mice, HBV-infected patients, or human primary PBL cultures (data not shown). A significant body of work was done with infectious disease antigens that demonstrated that immunogenicity could be predicted on the basis of binding affinity of ≦500 nM (Sette, 1994a; Wentworth, 1996, Alexander, 1997) and supertype cross-reactivity (Threlkeld, 1997; Bertoni, 1997; Doolan, 1997; Scognamiglio, 1999). A correlation between binding affinity and immunogenicity was also demonstrated for HLA-A2-restricted TAA (Keogh, et al. J. Immunol. 167(2):787-91 (2001)). In the case of TAA-derived wildtype peptides, binding affinity ≦200 nM and supertype cross-reactivity were highly predictive of endogenous recognition (75%). The same success rate was achieved with analogs when primary immunogenicity was considered in addition to binding affinity and supertype binding.

Based on this knowledge, the principal strategy for epitope identification has been to first identify candidate peptides in the wildtype antigen sequence by their MHC binding motif, then to determine their binding affinity and supertype crossreactivity (DiBrino, 1993; Sette, 1994b). High affinity, crossreactive peptides are then tested for in vitro immunogenicity with PBMCs from normal donors and their ability to induce tumor-reactive CTLs (Celis, 1994a; Celis, 1994b; Kawashima, 1998, Feltkamp, 1994).

An extension of this strategy adopted to identify peptides for potential inclusion in vaccines is the development of primary anchor-substituted analogs (Ruppert, 1993). This strategy involves the identification of peptides carrying suboptimal residues at their primary anchor positions, and the replacement of one or more of these suboptimal residues with optimal anchor residues to enhance binding affinity to the predominant allele of the superfamily and/or crossreactivity to the other alleles in the superfamily. This strategy has been used to generate analogs of A2 restricted peptides (Keogh, et al. J. Immunol. 167(2):787-96 (2001)). The preferred and tolerated amino acids at each anchor position for A3, A1, A24 and B7 binding peptides have been identified in order to formulate analoging strategies for these alleles. The results of those efforts are briefly summarized herein and in Sidney, et al. Hum. Immunol. 62(11):1200-16 (2001).

The use of analogs is also relevant to address the problem of expanding the number of potential epitopes of a given tumor antigen, particularly in the case of small proteins such as p53. In addition, broadly crossreactive supertype binding analogs increase population coverage of a given epitope. Analogs can also be used to enhance the immunogenicity of known epitopes. Another advantage is to increase peptide manufacturability and stability by substituting, for instance, α-aminobutyric acid (B) for cysteine (Sette, Persistent Viral Infections, review).

Sarobe (1998), Vierboom (1998) and Irvine (1999) demonstrated that A2-restricted, anchor-analoged epitopes derived from TAA and infectious disease antigens showed improved immunogenicity in mice. Other investigators have demonstrated (Rosenberg, 1998; Zaremba, 1997) that when analogs with binding better than the corresponding wildtype peptide were used to stimulate cells from cancer patients in vitro, a peptide-specific CTL response was detected after far fewer restimulations than were required with the wildtype peptide (Rosenberg, 1998; Zaremba, 1997). Most importantly, tumor killing was also observed. Based on these results, a much stronger CTL response would be anticipated in vivo. Additionally, in a clinical trial, Rosenberg et al (1998) have observed tumor regression with a melanoma analog in conjunction with IL2 therapy, demonstrating the value of analog peptides as immunotherapeutics. Similar results were obtained with fixed anchor analogs and PBMCs from normal donors (data not shown). Those results clearly demonstrated that these peptides are strong immunogens capable of generating wildtype peptide and tumor cell reactive CTLs.

There has been little information in the literature describing TAA-derived epitopes for the non-A2 alleles (A3, B7, A1 and A24). Our strategy is to identify novel peptide epitopes that demonstrate high HLA-A*03, -B*07, -A*01 or -A*24 binding affinity and supertype binding where applicable in order to elicit a strong CTL response. Kawashima (1998); Castelli (1998), Kittlesen (1998), Tahara (1999) and others have demonstrated CTL responses to tumor epitopes in normal donors or cancer patients, which would indicate that tolerance is incomplete. It appears that immune tolerance at the CTL level does not completely eliminate or inactivate CTL precursors capable of recognizing high affinity class I peptides.

Here we report the identification of HLA-A3, -A1, -A24 and -B7-restricted vaccine candidate peptides from the CEA, MAGE2/3, HER2/neu and p53 tumor antigens. Candidates selected for in vitro immunogenicity assays on the basis of their HLA affinity and supertype cross-reactivity were also demonstrated to be immunogenic and capable of inducing CTL that recognize tumor target cells.

Materials and Methods

MHC Sources

The following EBV-transformed cell lines were used as sources of class I major histocompatibility complex molecules: Steinlin (A*0101), AMAI (B*5301), GM3107 (A*0301, B*0702), BVR (A*1101), SPACH (A*3101), LWAGS (A*3301), KAS116 (B*51), and KT3 (A*2402, B*5401). A ClR transfectant was used for the isolation of A*6801, as well as for B*3501. These C1R transfectants were characterized by Dr. Walter Storkus and Dr. Masafumi Takaguchi, respectively.

Cells were maintained in vitro by culture in RPMI 1640 medium supplemented with 2 mM L-glutamine and 10% heat-inactivated FCS. Cells were also supplemented with 100 μg/ml of streptomycin [Irvine Scientific, Santa Ana, Calif.] and 100 U/ml of penicillin [Life Technologies, Carlsbad, Calif.]. Large quantities of cells were grown in spinner cultures.

Affinity Purification of HLA-A and -B Molecules.

Cells were lysed at a concentration of 10⁸ cells/ml in PBS containing 1% NP-40 and 1 mM PMSF. The lysates were cleared of debris and nuclei by centifugation at 10,000×g for 20 min.

MHC molecules were then purified by affinity chromatography as previously described (Sette, 1998; Ruppert, 1993). Columns of inactivated Sepharose CL4B and Protein A Sepharose were used as pre-columns. Lysates were filtered through 0.8 and 0.4 μM filters and then depleted of HLA-B and HLA-C molecules by repeated passage over Protein A Sepharose beads conjugated with the anti-HLA (B,C) antibody B1.23.2. Typically 2 to 4 passages were required for effective depletion Subsequently, the anti-HLA (A,B,C) antibody W6/32 was used to capture HLA-A molecules.

Independently, both antibody columns were washed with 15-column volumes of 10 mM TRIS in 1.0% NP-40, PBS and 2-column volumes of PBS containing 0.4% n-octylglucoside. Finally, the class I molecules were eluted with 50 mM diethylamine in 0.15M NaCl containing 0.4% n-octylglucoside, pH 11.5. A 1/25 volume of 2.0M Tris, pH 6.8, was added to the eluate to reduce the pH to 8.0, and then concentrated by centrifugation in Centriprep 30 concentrators (Amicon, Beverly, Mass.) at 200 rpm. Protein purity, concentration, and effectiveness of depletion steps were monitored by SDS-PAGE and BCA protein analysis (Sigma).

Class I Peptide-Binding Assays.

Purified human class I molecules [5 to 500 nM] were incubated with 1-10 nM ¹²⁵I-radiolabeled probe peptide, iodinated by the Chloramine T method (Buus, 1987), for 48 h at room temperature in the presence of 1 μM human β2M (Scripps Laboratories, San Diego, Calif.) and a cocktail of protease inhibitors. The final concentrations of protease inhibitors were: 1 mM PMSF, 1.3 nM 1.10 phenanthroline, 73 μM pepstatin A, 8 mM EDTA, and 200 μM N alpha-tosyl-lysine chloromethyl ketone (TLCK).

Class I peptide complexes were separated from free peptide by gel filtration on TSK200 columns, and the fraction of bound peptide calculated as previously described (Sette, 1998). In preliminary experiments, the HLA class I preparation was titered in the presence of fixed amounts of radiolabeled peptides to determine the concentration of class I molecules necessary to bind 10-20% of the total radioactivity. All subsequent inhibition and direct binding assays were then performed using these class I concentrations. In the inhibition assays, peptide inhibitors were typically tested at concentrations ranging from 120 μg/ml to 1.2 ng/ml. The data were then plotted and the dose yielding 50% inhibition was measured. Peptides were tested in two to four completely independent experiments. Since under these conditions [label]<[MHC] and IC₅₀≦[MHC], the measured IC₅₀s are reasonable approximations of the true k_(D) values.

Radiolabeled probe and standard control peptides used are as follows:

An A3 non-natural consensus peptide (A3con; sequence KVFPYALINK) (SEQ ID NO: 747) (Sette 1994b; Kubo, 1994) was used as the radiolabeled probe for the A3, A11, A31, and A*6801 assays. An HBV 141.Y7 analog of HBVc 141-151 (sequence STLPETYVVRR) (SEQ ID NO: 748) was used as the radiolabeled probe for the A*3301 assay. The average IC₅₀'s of A3con for the A3, A11, A31, and A*6801 assays were 11 nM, 6.0 nM, 18 nM, and 8.0 nM, respectively. The average IC₅₀ of the HBVc 141-151 peptide in the A*3301 assay was 29 nM.

B35con2 (sequence FPFKYAAAF) (SEQ ID NO: 749) was used as the radiolabeled probe and standard control peptide for the B*3501, B*5101, B*5301, and B*5401 assays. The IC₅₀'s of B35con2 for each of these assays were 7.2 nM, 5.5 nM, 9.3 nM, and 10 nM, respectively. The A*0201 Signal Sequence 5-13a.Y7 (APRTLVYLL) (SEQ ID NO: 750) (Huczko, 1993; Chen, 1994; Sidney, 1996a,b) was used as the radiolabeled probe and standard peptide for B*0702 assay. It had an average IC₅₀ of 5.5 nM.

The human J chain peptide (sequence YTAVVPLVY) (SEQ ID NO: 751) was used as the radiolabeled probe for the A1 assay, utilizing an A1 con peptide (sequence YLEPALIKY) (SEQ ID NO: 752) as the standard control. It had an average IC₅₀ of 25 nM. The A24con peptide (sequence AYIDNYNKF) (SEQ ID NO: 753) was used as the radiolabeled probe and standard control peptide for the A24 assay, having an average IC₅₀ of 12 nM.

Peptide Synthesis.

Peptides were either synthesized at Epimmune, Inc. (San Diego, Calif.), as previously described (Sette, 1998), or, for large epitope libraries, purchased as crude material from Chiron Technologies Corp (Clayton, Victoria, Australia). Peptides synthesized at Epimmune were purified to >95% homogeneity by reverse-phase HPLC. The purity of these synthetic peptides was determined on an analytical reverse-phase column and their composition ascertained by amino acid analysis and/or mass spectrometry analysis.

Identification of Motif-Positive Peptides.

HLA-A3 Supertype

Protein sequences from the targeted four tumor antigens (p53, CEA, HER2/neu, and MAGE2/3) were scanned, utilizing a customized program, to identify 8-, 9-, 10-, and 11-mer sequences containing the HLA-A3 supertype main anchor motif. That motif is leucine (L), isoleucine (1), valine (V), methionine (M), alanine (A), serine (S) or threonine (T) at position 2, and either lysine K) or arginine (R) at the C-terminus.

Naturally-occurring, or wild-type (WT) peptides identified as described above were tested for their capacity to bind purified HLA-A*0301 and A*1101 molecules in vitro. Peptides exhibiting high (IC₅₀≦50 nM) or intermediate binding affinity (IC₅₀≦51-500 nM) for either one or both of these primary A3 supertype alleles were then tested on other predominant molecules of the A3 supertype family (A*3101, A*3301, and A*6801) (Sidney, 1996a). Peptides binding at least three of the five alleles tested were classified as “crossbinders”, and candidates for cellular screening analysis. The rationale for having co-primary alleles for the A3 supertype is based on similar peptide motifs as well as the dichotomy of these alleles' population coverage. A*0301 has the highest phenotypic frequencies for Caucasians and North American blacks, and A*1101 has the best coverage for Asians.

HLA-B7 Supertype

The targeted TAA sequences were scanned to identify 8-, 9-, 10-, and 11-mer sequences containing, proline (P) at position 2, and L, I, V, M, A, phenylalanine (F), tryptophan (W), or tyrosine (Y) at the C-terminus (HLA-B7 supertype motif).

These peptides were tested for their capacity to bind purified HLA-B*0702 molecules in vitro. Peptides exhibiting high or intermediate binding affinity were then tested on other predominant molecules of the B7 supertype family (B*3501, B*5101, B*5301, and B*5401) (Sidney, 1995). Peptides that are B7 supertype crossbinders were candidates for cellular screening analysis.

HLA-A1 and HLA-A24 Motifs

For A24 sequences, peptides carrying Y, F, W, or M at position 2, and F, I, L, or W at the C terminus were identified as motif positive. The A1 motif is somewhat unique in that it possesses a dual primary anchor motif; one at position 2 and the C terminus, another at position 3 and the C terminus (Kubo, 1994; Kondo, 1997). The residues associated with the A1 motif are T, S, or M at position 2, aspartic acid (D), glutamic acid (E), A, or S at position 3, and Y at the C terminus.

Peptides were tested for their capacity to bind the appropriate purified HLA-A1 and A24 motif carrying molecule in vitro.

Definition of Distinct Binding Regions

When a protein sequence is scanned for the presence of motif-positive peptides, it is not uncommon for several peptides to be identified within a few residues of each other. Often times, a 9-mer peptide is nested within a 10- or 11-mer sequence. It is possible that some overlap exists amongst responses elicited by such nested or overlapping epitopes, and we thereby consider such epitopes as closely related. For vaccine development, we generally recommend inclusion of only one epitope from each such family of overlapping peptides. On the other hand, we define distinct regions as peptides having a first position at least 4 residues apart. It is safe to assume that upon binding to the MHC, these peptides would induce responses from different T cell receptors (TCR), and should therefore be considered as distinct for the purpose of epitope selection.

Primary Anchor Position Residue Substitution Strategy.

It has been shown that class I peptide ligands can be modified to increase their binding affinity and/or degeneracy (Sidney, 1996b; Rosenberg, 1998). More importantly, modified peptides have also been shown to possess increased immunogenicity and crossreactive recognition by T cells specific for the WT epitope (Parkhurst, 1996; Pogue, 1995). This modification, sometimes referred to as “fixing”, entails analoging peptides by replacing sub-optimal amino acids at primary anchor positions for optimal residues. What residues are optimal is dependent upon the allele under examination. This strategy was successfully employed in the A2 system as part of an effort to develop an A2 therapautic cancer vaccine (data not shown).

For the various supertypes and alleles addressed in this study, motifs are listed in Table 2. HLA binding data from peptides selected using these motifs was analyzed to develop a strategy for analoging TAA-derived peptides with suboptimal anchor residues at anchor positions.

For the A3 supertype, residues L, L M, A, or S are suboptimal at position 2 and can be substituted with T or V. At the C terminus, both R and K are canonical residues, each displaying a propensity towards specific alleles within the A3 supertype with lysine preferred by A*0301 and A*1101, arginine is preferred by A*3101, A*3301, and A*6801.

For the B7 supertype, proline is absolutely required at position 2 and therefore only the C terminus is a candidate for analoging in terms of primary anchors. Residues L, M, A, V, F, W, or Y are suboptimal, and can be substituted with I. Additionally, we have observed that the presence of a bulky aromatic, specifically phenylalanine (F), at position 1 of a B7 supermotif peptide can significantly increase B7 binding and crossbinding capacity (Table 15).

For the A24 motif, residues F, W, or M at position 2 are suboptimal and should be substituted with Y. At the C terminus, I, L, or W are suboptimal and should be substituted with F.

The A1 allele is associated with a dual primary anchor motif (or 2 submotifs). For one submotif, T is preferred over S and M at position 2. The second submotif prefers D over E, A, and S at position 3. Additionally, Y is the optimal residue at the C terminus for both submotifs, but substituting for A, F, or W if both T₂ and D₃ are present is a viable alternative.

In addition, WT peptide candidates for analoging must exhibit at least weak binding (IC₅₀ of ≦5000 nM) to the parent allele (A1 or A24), or weakly bind at least 3 of 5 alleles (A3 and B7 supertypes). The rationale for this requirement is that the WT peptide must be present endogenously in sufficient quantity to be biologically relevant.

Another analog utilized in these studies, unrelated to the primary anchor position, involves the substitution of α-amino butyric acid (B) for cysteine (C). Due to its chemical nature, cysteine has the propensity to form disulfide bridges and sufficiently alter the peptide structurally so as to reduce binding capacity. Substituting B for C not only alleviates this problem, but has been shown to actually improve binding and crossbinding capability in certain instances (Review: Sette, Persistent Viral Infections, Ed. R. Ahmed and I. Chen, John Wiley & Sons, England).

Target Cell Lines for HLA-A3 and -B7 Cellular Screening

The Epstein-Barr transformed homozygous cell lines EHM (A3+, ASHI cell repository, currently inactive) or GM3107 (A3+, B7+; Human Genetic Mutant Repository) were used as the peptide-loaded target to measure activity of HLA-A3-restricted CTL. The JY cell line (B7+, a gift from L. Sherman at The Scripps Research Institute) was used as the peptide-loaded target cells to measure activity of HLA-B7-restricted CTL. The negative and positive tumor target cell lines used for each antigen were: SW480 (A3-, CEA+) (ATCC No. CCL-228) and SW403 (A3+, CEA+) (ATCC No. CCL-230) for CEA; SW480 (A3-, HER2/neu+) and SW403 (A3+, HER2/neu+) for HER2/neu; 938mel (A3-, MAGE2/3+) and 624mel (A3+, MAGE2/3+) for MAGE2/3; and SW403 (A3+, p53-) and SW403 transfected with p53 (A3+, p53+) for p53. The HLA-typed melanoma cell lines (624mel and 938mel) were a generous gift from Y. Kawakami and S. Rosenberg, National Cancer Institute, Bethesda, Md. The tumor cell lines, SW403 and SW480 were obtained from the American Type Culture Collection (Manassas, Va.). The EBV transformed and melanoma cell lines were grown in RPMI-1640 medium supplemented with antibiotics, sodium pyruvate, nonessential amino acids and 10% (v/v) heat inactivated FCS. SW403 and SW480 were grown in DMEM with the same additives. The tumor target cells were treated with 100 U/ml IFNγ (Genzyme) for 48 hours at 37° C. prior to use as targets in the ⁵¹Cr release and in situ IFNγ assays.

Primary CTL Induction Cultures

Generation of dendritic cells (IC): Monocytes were purified from previously frozen PBMCs by plating 10×10⁶ cells in 3 ml of complete medium (RPMI with 5% heat-inactivated human AB serum, penicillin, streptomycin, sodium pyruvate and non-essential amino acids) in each well of a 6-well plate. After 2 hrs at 37° C., the non-adherent cells were removed and three ml of complete medium containing 50 ng/ml of human rGM-CSF and 1,000 U/ml of human rIL-4 were then added to each well. On day 6, the non-adherent cells were harvested, washed and cultured at 0.3-0.5×10⁶ cells/ml in complete medium with 75 ng/ml TNFα (R&D Systems).

On day 8, the DC were collected, washed, and pulsed with 40 μg/ml of peptide at a cell concentration of 1-2×10⁶/ml in the presence of 3 μg/ml P₂ microglobulin for 4 hours at 20° C. The DC were then irradiated (4,200 rads), washed 1 time with medium and counted again.

Induction of CTL with DC and Peptide: CD8+ T-cells were isolated by positive selection with Dynal immunomagnetic beads and detachabead reagent according to the manufacturer's instructions. Typically 200˜250×10⁶ PBMC were processed to obtain 24×10⁶ CD8+ T cells (enough for a 48-well plate). 0.25 ml of CD8+ T-cells (@ 2×10⁶ cell/ml) were co-cultured with

0.25 ml cytokine-generated DC (@1×10⁵ cells/ml) in each well of a 48-well plate in the presence of 10 ng/ml human rIL-7 (Endogen). Human rIL10 (Endogen) was added the next day at a final concentration of 10 ng/ml and human rIL2 was added on day 2 at 10 IU/ml.

Restimulation of the induction cultures with peptide-pulsed adherent cells: Seven and fourteen days after the primary induction, the cells were restimulated with irradiated, peptide-pulsed adherent cells. Briefly, adherent cells were pulsed with 10 μg/ml of peptide in the presence of 3 μg/ml β₂ microglobulin in RPMI/5% human AB serum for 2 hours at 37° C. The wells were washed once with RPMI. Most of the medium was aspirated from the induction cultures (CD8+ cells) and brought to 0.5 ml with fresh medium and the cells were transferred to the wells containing the peptide-pulsed adherent cells. Human rIL10 was added at a final concentration of 10 ng/ml 24 hours later and human rIL2 was added after 48 hours and again 2-3 days later at 50 IU/ml (Tsai, 1998). Seven days after the second restimulation, the cultures were assayed for CTL activity in a ⁵¹Cr release assay or in situ IFNγ ELISA.

Measurement of CTL Lytic Activity by ⁵¹Cr Release

Cytotoxicity was determined in a standard ⁵¹Cr release assay by assaying individual wells at a single E:T ratio. Peptide-pulsed targets were prepared by incubating the cells with 10 μg/ml peptide overnight at 37° C. Adherent target cells were removed from culture flasks with typsin-EDTA. Target cells were labeled with 200 μCi of ⁵¹Cr sodium chromate (Dupont, Wilmington, Del.) for 1 hour at 37° C., washed twice, resuspended at 10⁶ per ml and diluted 1:10 with K562 cells (an NK-sensitive erythroblastoma cell line used to reduce non-specific lysis) at a concentration of 3.3×10⁶/ml. Target cells (100 μl) and 100 μl of effectors were plated in 96 well round-bottom plates and incubated for 5 hours at 37° C. 100 μl of supernatant were collected from each well and percent lysis was determine according to the formula: [(cpm of the test sample−cpm of the spontaneous ⁵¹Cr release sample)/(cpm of the maximal ⁵¹Cr release sample−cpm of the spontaneous ⁵¹Cr release sample)]×100. Maximum and spontaneous release was determined by incubating the labeled targets with 1% Triton X-100 and medium alone, respectively. A positive culture was defined as one in which the specific lysis (sample—background) was 10% or higher in the case of individual wells and was 15% or more at the 2 highest E:T ratios when expanded cultures were assayed.

In Situ Measurement of Human IFNγ Production

In brief, Costar EIA plates were coated with mouse anti-human IFNγ monoclonal antibody (Pharmingen) overnight at 4° C. The plates were washed and blocked for 2 hours, after which the CTLs (100 μl/well) and targets (100 μl/well) were added to each well, leaving empty wells for the standards and blanks (which received medium only). For expanded cultures, 1×10⁵ CTL/well were mixed with 1×10⁵ targets (neg. control) or peptide-pulsed or endogenous targets. All wells were brought to 2001 μl with medium and incubated for 48 hours at 37° C. with 5% CO₂.

Human rIFNγ (R&D Systems) was added to the standard wells starting at 400 pg/100 ul/well and the plate incubated for 2 hours at 37° C. The plates were washed and 100 μl biotinylated mouse anti-human IFNγ monoclonal antibody (Pharmingen) were added to each well and the plates incubated for 2 hours at room temp. After washing again, 100 μl/well HRP-streptavidin (Zymed) were added and incubated for 1 hour at room temp. The plates were then washed 6× with wash buffer, 100 μl/well TMB developing solution (KPL, mixed 1:1) was added and the plates allowed to develop for 5-15 min. The reaction was stopped with 50 μl/well 1 M H₃PO₄ and read at OD450. A culture was considered positive if it measured at least 50 pg of IFNγ/well above background and was at least twice the background level of expression.

CTL Expansion

Those cultures that demonstrated activity against peptide-pulsed targets and/or tumor targets were expanded over a two week period with anti-CD3 antibodies (Wang, 1998; Greenberg, 1991). Briefly, 5×10⁴ CD8+ cells were added to a T25 flask containing the following: 1×10⁶ irradiated (4,200 rad) PBMC (autologous or allogeneic) per ml, 2×10⁵ irradiated (8,000 rad) EBV-transformed cells per ml, and OKT3 at 30 ng per ml in RPMI-1640 containing 10% (v/v) human AB serum, non-essential AA, sodium pyruvate, 25 μM 2-ME, L-glutamine and penicillin/streptomycin. Human rIL2 was added 24 hours later at a final concentration of 200 IU/ml and every 3-4 days thereafter with fresh medium at 50 IU/ml. The cells were split if the concentration exceeded 1×10⁶/ml and the cultures assayed between days 13 and 15.

Alternatively, cultures were expanded by stimulation with peptide-pulsed autologous PBMCs in the absence of OKT3. All other conditions and cell numbers remained the same as those described above.

Results:

Scanning for Motif-Positive Peptides

As described in the Materials and Methods section, protein sequences from the four tumor antigen targets (p53, CEA, Her2/neu, and MAGE2/3) were scanned, utilizing a customized program, for 8-, 9-, 10-, and 11-mer sequences containing one of four motif types. Specifically, peptides carrying motifs of the HLA-A3 supertype, the HLA-B7 supertype, the HLA-A24 motif, and the HLA-A1 motif were identified. The specific residues associated with each of these motifs or supermotifs are listed in the Materials and Methods section. For the HER2/neu antigen, only the intracellular domain was examined.

A number of naturally occurring, or wildtype (WT) peptides identified utilizing these motifs are listed in Tables 6, 9, 10, 16a-d, 17a-d, 18a-d and 19a-d. Also presented is the number of analogs that could be synthesized. In general, WT peptides carrying suboptimal residues at primary anchor positions can be substituted with optimal residues, employing the analog strategies described in the Materials and Methods section. To be considered a candidate for analoging, a TAA-derived WT peptide must carry one preferred residue at either position 2 or the C terminus. In previous work in the A2 system, analogs tested for immunogenicity which had residues substituted at both anchor positions were able to induce peptide-specific CTLs in vitro. However, those CTLs were able to recognize tumor cell targets only 40% of the time as compared to 75% for analogs with a single anchor position substitution, and therefore were not pursued in the A3, B7, A1 and A24 systems. For the B7 supermotif-positive peptides, another analog strategy was discovered from previous unrelated studies that substituting the position 1 residue with phenylalanine (F) greatly improved B7 crossbinding capability. These analogs are also included in Table 15 and Table 17a-d. However, these F1 analogs will not be considered as candidates until this strategy has been validated. That is, induction of tumor-reactive CTLs must be demonstrated with these analogs.

Additionally, Table 15 presents, for each TAA, the number of WT peptides and analogs actually synthesized and tested for supertype or primary allele binding affinity. As seen, the vast majority of WT peptides have been tested for binding affinity. The synthesis and testing of analogs, however, are in various stages of completion dependent upon the TAA. Recall that for an analog to be a candidate for immunogenicity screening, its WT parent peptide must bind at least weakly, ie. IC₅₀≦5000 nM, to 3 of 5 alleles in the supertype, or to the parent allele in the cases of A1 and A24. The numbers presented in the analog, motif-positive columns are based solely on analoging potential, or the presence of suboptimal residues at anchor positions, and not on measured binding. For this reason, many of the analogs listed would not ultimately be synthesized.

To date, 695 CEA-derived motif positive peptides have been identified and 358 of these tested for supertype or primary binding. These include 64 A3 binding wildtype and analog peptides, 171 B7 peptides, 65 A1 peptides and 28 A24 peptides. Analysis of the intracellular domain of the HER2/neu protein identified 822 motif positive peptides. Of these, 364 (64 A3, 213 B7, 81 A1 and 47 A24) have been tested to determine their binding affinities. The MAGE2 and MAGE3 proteins were considered together and a total of 611 motif positive peptides were identified. A subset of these, including 80 A3 wildtype and analog peptides, 146 B7 peptides, 40 A1 peptides and 54 A24 peptides were tested for primary and/or supertype binding. Lastly, 566 motif positive, p53-derived peptides have been identified. The wildtype and analog peptides tested to determine their binding affinity included 102 A3-restricted peptides, 165 B7-restricted peptides, 19 A1-restricted peptides and 14 A24-restricted peptides. The results of the binding assays on these motif positive peptides and the subsequent identification of epitopes is the subject of this report.

Analysis of HLA-A3 Vaccine Candidates

CEA: A total of 64 CEA-derived peptides were tested for binding to A3 supertype molecules to date. Fourteen CEA-derived peptides from 8 distinct regions of the protein sequence have been identified. These fourteen peptides have a binding affinity of ≦500 nM to A*0301 and/or A*1101, the two most predominant alleles, and also crossbind to a total of at least 3 alleles of the A3 superfamily (Table 16a).

All fourteen bind HLA-A*1101 with high or intermediate affinity. Noteworthy is the case of the CEA.61 epitope that binds with high affinity (≦50 nM) to all five alleles, is immunogenic and is generated by natural processing. High A*1101 binding affinity has also been observed in 5 WT peptides of the remaining six regions: CEA.376, CEA.418, CEA.419, CEA.554 and CEA.636. Three of these, CEA.419, CEA.554 and CEA.636, bind to an additional three alleles and are also good candidate peptides.

Five of the seven regions exhibited suboptimal A*0301 binding for all of the WT peptides encompassed. Peptides from three of these regions were analoged in an attempt to improve A3 binding and overall crossbinding capacity. The CEA.241K10 analog displayed a 26-fold improvement in A3 binding. This analog was demonstrated to be immunogenic in izl vitro immunogenicity assays, and the resulting CTLs were shown to be cross-reactive with target cells pulsed with the wildtype peptide (FIG. 1). A second analog within the same region, CEA.241V2, was associated with improved binding to A*0301 and A*1101, and bound 4 of 5 superfamily alleles. Therefore, this peptide is also a strong candidate. Significant increases were observed with CEA.420V2, which exhibited the most suitable binding profile of any peptide derived from that region, which included 3 different wild-type sequences. Lastly, CEA.376V2 and CEA.554V2 showed no improvement in binding capacity over their wildtype counterparts.

In conclusion, within the eight distinct regions of CEA identified to harbor motif positive peptide candidates, multiple peptides have been identified which bind each of the five supertype alleles. Two peptides bind all five alleles and another six bind four of the five. A vaccine could be designed with one peptide from each region, which would include at least 4 peptides specific for each allele of the A3 superfamily.

HER2/neu: When motif analysis was performed on the intracellular and transmembrane domains of the 1255 amino acid HER/neu protein sequence and binding affinity of the motif positive peptides determined, candidate peptides from 11 different regions were identified (Table 16b). A total of sixteen wild-type peptides and analogs demonstrated binding affinities of ≦500 nM to at least 3 alleles in the A3 superfamily. Eleven of these bound HLA-A*0301 and fifteen bound A*1101, and 10 peptides had a binding affinity of ≦500 nM for both alleles. The HER2/neu.681 epitope was also considered here because primary CTL data indicates that this peptide was immunogenic and has been shown to induce tumor-reactive CTLs. Three additional peptides, HER2/neu.754 (Kawashima, 1999), HER2/neu.669 (9-mer), and HER2/neu.852 (Kawashima, 1999), bind with an affinity ≦500 nM to 24 alleles and are immunogenic. Five additional WT peptides (HER2/neu.806, HER2/neu.846, HER2/neu.889, HER2/neu.972 and HER2/neu.997) also demonstrated high and intermediate binding to 2 alleles and therefore are candidate peptides.

Of the six analog peptides tested, 2 showed improvement as compared to their wildtype peptide counterparts. More specifically, HER2/neu.860V2 improved binding affinity to A*6801, while HER2/neu.889V2 exhibited improved binding to HLA-A*0301 and A*6801.

For each of the five supertype alleles, multiple peptides have been identified which bound with affinities of 500 nM or less. Two peptides bind all five alleles and six additional candidates bind 4 alleles. In total, six of the ten regions have peptides that bind ≧4 of 5 alleles representing a substantial pool of A3 supertype peptides that could be considered for use in a vaccine.

MAGE2/3: Motif analysis of the MAGE 2/3 protein sequences (each 314 amino acids long) identified 22 peptides from 9 distinct regions which are high or intermediate cross-reactive binders of the A3 superfamily (Table 16c). More specifically, sixteen of these bind both A*0301 and A*1101. MAGE2.73 binds 5 alleles and induced CTL capable of recognizing both peptide-pulsed and tumor targets (Epimmune U.S. Pat. No. 6,037,135). MAGE2.237, MAGE2.277 and MAGE3.189 bind to 3 of the 5 alleles. Interestingly, Reynolds and co-workers (Reynolds et al., J. Immunol. Methods 244:59-67 (2000) have demonstrated in an ELISPOT assay that MAGE3.189 is antigenic (eg. when melanoma patients were given a vaccine comprised of supernatant from a melanoma cell line, CTLs could be isolated that recognized MAGE3.189-pulsed target cells).

It can also be noted that the MAGE2.226 and MAGE3.226 peptides are homologous except for a M to V difference at position 2. Since the main MHC anchor residues are least likely to influence T cell receptor recognition (Zhang, 1992), these peptides are likely to induce overlapping T cell specificity and therefore could be considered variations of the same epitopic sequence. Of the two, MAGE3.226 is preferred at this point because it binds 2 of the 5 alleles with higher affinity. In particular, this epitope binds HLA-A*3301 more than 10-fold better than the K9 analog, which binds all 5 alleles but with significantly lower A*3301 affinity.

Additional analogs were generated with improved binding characteristics. Specifically, MAGE2.69K9 showed improved cross-reactivity in regard to A*0301, A*1101 and A*6801 binding. MAGE2.299V2 demonstrated improved A*0301 binding and introducing V at position 2 in MAGE3.138 significantly improved the A*0301, A*1101 and A*6801 binding affinities. Substituting K or R at position 9 of the MAGE3.116 peptide improved crossreactivity to 3 and 5 alleles, respectively.

For each of the five supertype alleles, multiple peptides have been identified which bound with affinities of 500 nM or less. Three peptides bound all five alleles, one candidate bound 4 alleles and an additional six peptides bound 3 alleles. In total, all nine regions have peptides that bound ≧3 of 5 alleles.

p53: A total of 17 high and intermediate affinity cross-reactive motif positive peptides, 7 wildtype and 10 analogs, were identified as a result of scanning the 393 amino acid p53 protein sequence (Table 16d). These peptides are derived from 8 different regions of the protein. All seventeen bind A*0301 and 16/17 bind A*1101. Several of the wildtype peptides are potential candidate peptides. The p53.124 peptide binds 3 alleles and provides coverage of A*6801. The 8-mer of p53.273 binds 4 out of 5 alleles, including A*3101 and A*3301. p53.132 and p53.376 bind 3 alleles each but bind only A*0301 and A*1101 with high affinity.

The p53.101, p53.172 and two p53.240 peptides could also represent candidates for inclusion in an epitope based vaccine, depending on supertype binding results.

Six of the eight regions described have had WT peptides analoged. Two of these analogs, p53.172B5K10 and p53.240B3K9, demonstrated improved binding to one of the two primary alleles (A*0301 or A*1101). Additionally, the p53.172B5K10 epitope bound 3 alleles of the A3 superfamily, is immunogenic and able to induce CTLs that recognize both the wildtype sequence and p53 transfected tumor targets (FIG. 2). The p53.240V2B3 is also a good candidate for inclusion in a multi-epitope vaccine based on the binding affinities observed, pending WT supertype binding results.

With regard to results generated for the other analogs, p53.156R9 showed improved binding to A*3301, an allele for which few p53-derived wildtype peptides bound. Finally, the p53.101K10 and p53.101V2 peptides are similar with respect to binding affinity and allelic coverage but immunogenicity has been demonstrated for the K10 analog (data not shown) making it a stronger candidate than the V2 analog.

Supertype crossreactive candidates from all eight protein regions have been identified and all the candidates described above bind 3 to 4 of the supertype alleles. Coverage of all five alleles can be achieved by including multiple epitopes in a vaccine.

Analysis of HLA-B7 Vaccine Candidates

HLA-B7 supertype peptides are identified by their binding affinity for B*0702, the primary allele of the B7 superfamily, and two or more additional alleles. The overall number of B7 cross-reactive candidates is limited primarily because proline is the only tolerated amino acid at primary anchor position 2 while other supertypes (eg. A2 and A3) tolerate several different amino acids at their anchor positions (Sidney, 1995; 1996b). This restriction also limits the number of analogs that can be generated for a particular peptide.

Three regions of the CEA protein sequence are represented by 3 candidate peptides, one wildtype and 2 analogs (with isoleucine substituted at the C terminus), which bind B*0702 and 2-3 additional alleles with an affinity ≦500 nM (Table 17a).

Three wildtype peptides derived from 3 regions encoded within the intracellular domain of HER2/neu have also been identified (Table 17b). Substituting I for A at position 8 of the HER2/neu.921 peptide increases the binding affinities for B*0702 and B*5101 and therefore the analog would be considered a preferred candidate of that region. The 4 candidate peptides listed in Table 17b bound 3 to 4 alleles of the B7 supertype with an affinity ≦500 nM.

Motif analysis of the MAGE2 and MAGE3 protein sequences yielded a number of candidates from four discrete regions of these antigens which bind to ≧3 alleles of the B7 superfamily with an affinity ≦500 nM (Table 17c). MAGE2.170 binds to 4 alleles of the superfamily and is capable of inducing peptide reactive CTLs in vitro. Three analogs (MAGE3.7119, MAGE3.7718 and MAGE3.196110) exhibit increased crossreactivity and Class I binding affinity.

Another factor to be considered in the selection of vaccine candidates is the potential relevance of using nested peptides. The MAGE3.196 peptides can be used to illustrate this point. The 10-mer, while not cross-reactive, has high binding affinity for B*5401. It also has the advantage of containing the 8- and 9-mer peptides within its sequence. In theory, the 10-mer would undergo trimming in the endoplasmic reticulum to the 8-mer and/or the 9-mer peptide (Paz, 1999), each of which binds other common alleles in the B7 superfamily. In addition, Reynolds et al. (submitted 1999), demonstrated antigenicity for the 9-mer wildtype peptide.

Lastly, two p53-derived peptides have been identified, p53.76 and p53.84 (Table 17d). The p53.76 11-mer bound 3 alleles with higher affinity than the 9-mer. While the p53.84 wildtype peptide bound with only intermediate affinity, this candidate would be an improved candidate than the corresponding analog because of higher binding affinities for B*5301 and B*5401.

In summary, three CEA-derived peptides from 3 distinct regions, 4 Her2/neu peptides from 3 regions, and 4 p53 peptides from two regions, all with binding affinities ≦500 nM and crossreactive for at least 3 supertype alleles, have been identified. An additional 10 MAGE2/3-derived high and intermediate affinity peptides from 4 regions were also identified.

Analysis of HLA-A1 Vaccine Candidates

CEA: Six high and intermediate binding CEA wildtype peptides from 6 regions of the 702 amino acid protein sequence have been identified (Table 18a). Results with other supertype alleles (eg. A2 and A3) indicate that peptides with an IC₅₀≦100 nM for the predominant allele of the superfamily also bound with high or intermediate affinity to the other alleles of the superfamily. Therefore, until binding assays are developed for the other alleles of the A1 superfamily, it is reasonable to employ the stricter 100 nM cut-off for the identification of HLA-A1-restricted, potentially degenerate binding peptides. Accordingly, four WT peptides are potential candidates based on a binding affinity of ≦100 nM to A*0101. These are CEA.225, CEA.403, CEA.581, and CEA.616.

CEA.616 and peptides from 4 additional regions were analoged at primary anchor positions, for 1 of the 2 submotifs, to improve coverage of this allele. The binding cut-off of the wildtype peptides to be analoged was 5000 nM to ensure expression of the tumor antigen-derived peptide on the tumor cell and the cut-off for the analog candidates was again ≦100 nM. Employing these criteria, an additional 5 analog peptides have been identified as candidates. The CEA.289D3, CEA.418D3, CEA.419D3, CEA.467D3, and CEA.616D3 peptides all demonstrate an HLA-A*0101 binding affinity ≦100 nM.

A total of 4 wildtype and 5 analog peptides derived from 7 distinct regions and with binding affinities ≦100 nM have been identified as candidates.

HER2/neu: The Her2/neu protein sequence was scanned to identify HLA-A1 motif positive peptides and the binding affinity of those peptides for HLA-A*0101 was determined. Wildtype peptides from 6 distinct regions were identified and 4 of these bind HLA-A*0101 with an affinity ≦100 nM (Table 18b). These are HER2/neu-826, HER2/neu.869, HER2/neu.899, and HER2/neu.1213.

All of the wildtype peptides were analoged at primary anchor positions to improve coverage of this antigen. Eleven analogs, representing a total of 9 regions, were identified as potential candidates based on a binding affinity ≦10 nM to HLA-A*0101. Of these, eight demonstrate significantly improved binding affinity. These are HER2/neu.773D3, HER2/neu.826T2, HER2/neu.996D3,HER2/neu.997T2, HER2/neu.1014T2, HER2/neu.1131D3, HER2/neu.1213T2, HER2/neu.1239D3.

In summary, sixteen wildtype and analog peptides representing 9 different protein regions meet the criteria for a vaccine candidate. That is, they bound to the primary allele of the A1 superfamily, HLA-A*0101 with an affinity of 100 nM or less.

MAGE2/3: Five HLA-A1 motif positive peptides with binding affinities ≦100 nM have been identified and are listed in Table 18c. These five wildtype peptides (MAGE3.246, MAGE2.247, MAGE3.68, MAGE3.166, and MAGE3.168) are from four non-overlapping regions of the two proteins. Additionally, Tuting et al (1998) demonstrated induction of MAGE3.168-specific CTLs as well as endogenous recognition of tumor targets expressing the epitope. Additionally, in clinical trials, tumor regression was observed in melanoma patients vaccinated with peptide-pulsed DC (Nestle et al, 1998) or the peptide alone (Marchand, 1999).

Eight wildtype peptides with a binding affinity between 501 and 5000 nM were analoged at primary anchor positions (Table 18c). Ten analogs, representative of 8 distinct regions, were identified as potential candidates. These are MAGE2.68D3, MAGE2.179D3, the T2 analogs of MAGE2.246/247, MAGE3.68D3, MAGE3.69T2, MAGE3.137T2, MAGE3.168T2, MAGE3.246D3 and MAGE3.293T2. Taken together, there are 15 peptides derived from 8 distinct regions of the MAGE2 and MAGE3 proteins.

p53: Four HLA-A1 motif positive wildtype peptides from 2 non-overlapping regions of the proteins were identified (Table 18d). These are p53.117, p53.225 (10-mer), p53.226 (9-mer) and p53.226 (1-mer), all of which bound HLA-A*0101 ≦100 nM. These are derived from 2 of the 4 regions shown in Table 18d.

Four wildtype peptides with an HLA-A*0101 binding affinity ≦5000 nM were analoged at primary anchor positions to improve coverage of this antigen (Table 18d). Four analogs, representing four distinct regions, were identified as potential candidates with an HLA-A*0101 binding affinity ≦100 nM. Two of these, the T2 substitution in p53.98 and the D3 substitution in p53.196, improved binding affinity more than 24-fold.

Binding analysis of the p53 wildtype and analog motif positive peptides has led to the identification of 8 candidates from four protein regions.

Analysis of HLA-A24 Vaccine Candidates

CEA: Twenty-one high and intermediate affinity HLA-A24 motif positive peptides, corresponding to 16 distinct regions of the CEA protein were identified (Table 19a). Only those peptides that bind with an affinity ≦100 nM were considered to be potential degenerate binders and therefore vaccine candidates. Using this criterion, nine CEA wildtype peptides from 9 non-redundant regions have been identified as potential candidate peptides. In addition, Nukaya et al (1999) demonstrated that CEA.268 induced CTLs in normal donors that recognized peptide-pulsed targets and HLA matched tumor targets. Kim et al (1998) obtained the same results with CEA.652. Both of these peptides are high affinity (≦100 nM) binders, further confirming the selection of vaccine candidates on the basis of their binding affinity.

Wildtype peptides from 10 regions were analoged at primary anchor residues to improve coverage of this allele (able 19a). The binding cut-off for the wildtype peptides to be analoged was 5000 nM to ensure expression of the tumor antigen-derived peptide on the tumor cell and the cut-off for the analog candidates was again ≦100 nM. Employing these criteria, eight additional peptides have been identified as candidates. Six of these analogs demonstrated binding affinities similar to their wildtype counterparts, while 2 analogs significantly improved binding to the HLA-A*2402 allele. These are CEA.446F10 and CEA.604F10.

Nine wildtype and eight analog peptides from 11 regions have been identified, providing an opportunity for wide coverage of this antigen.

HER2/neu: Seven wildtype peptides derived from 3 regions of the intracellular domain of HER2/neu were identified as HLA-A*2402 binders of high or intermediate affinity (Table 19b). Four of the seven peptides bound HLA-A*2402 ≦100 nM. These are HER2/neu.780, HER2/neu.907 and the 9- and 11-mers of HER2/neu.951.

Five wildtype peptides, with A*2402 binding affinities ≦5000 nM, were analoged at primary anchor positions to improve coverage of both this allele and tumor antigen. Four analogs were identified as potential vaccine candidates: HER2/neu.780F9, HER2/neu.907F9, HER2/neu.951F9 and HER2/neu.968Y2. The HER2/neu.968Y2 analog demonstrated the most significant increase (>18-fold) in binding affinity.

Eight peptides, 4 WT and 4 analogs, derived from 4 distinct regions of the intracellular domain of the HER2/neu protein have been identified as potential vaccine candidates.

MAGE2/3: Fifteen high and intermediate affinity HLA-A24 motif positive peptides, from 11 non-overlapping regions of these proteins, were identified (Table 19c). Eight of the 15 bound HLA-A*2402 ≦100 nM and represent 7 different regions of these proteins. Additionally, induction of CTLs and tumor target recognition were demonstrated for MAGE2.156 (Tahara, 1999) and MAGE3.195 (Tanaka, 1997).

Seven of the wildtype peptides were analoged at primary anchor residues to improve coverage of these antigens (Table 19c). Seven analogs, each representing a distinct region, were identified as potential candidate peptides, and three of these (MAGE2.97Y2, MAGE2.175F10 and MAGE3.175F10) demonstrated significantly higher binding affinity than the corresponding WT peptides. Overall, there are 15 wildtype and analog peptides that are derived from a total of 10 regions of the MAGE2 and MAGE3 proteins. Additionally, 2 of these peptides have been confirmed to induce peptide and tumor reactive CTLs by outside investigators.

p53: Five HLA-A24 motif positive peptides from 3 distinct regions have been identified (Table 19d). Two of these 5 bind HLA-A*0101 ≦100 nM. These are p53.102 (10-mer) and p53.125.

When the 10-mer of p53.102 was analoged at the C-terminus, a 3-fold improvement in binding affinity was observed. Analoging of p53.106 did increase the binding affinity 5-fold, but not to the imposed threshold of ≦100 nM. Overall, there are 3 p53-derived peptides (2 wildtype and 1 analog), representing two regions that are potential candidate peptides for a vaccine.

Binding to the Primary HLA-A3 and B7 Superfamily Alleles is Predictive of Degenerate Binding by TAA-Derived Peptides

Extensive analysis of infectious disease peptides demonstrated that a binding affinity ≦100 nM to the primary allele of a supertype was highly predictive of the degeneracy of a peptide. In other words, a majority of peptides meeting this criterion bound at least 2 other alleles of that superfamily with an affinity ≦500 nM (Bertoni, 1997; Doolan, 1997; Threlkeld, 1997). Based on this analysis, it is recommended that the 100 nM binding cut-off be used to select HLA-A*0101 and HLA-A*2402 peptide candidates in the absence of binding assays for the other supertype alleles. However, a significant number of HLA-A3 restricted TAA peptides were identified for the studies described here and supertype binding performed. This information was analyzed to determine if primary binding of ≦100 nM was predictive of supertype crossreactivity in the case of TAA-derived peptides. A total of 23 wildtype peptides (from Tables 16a-d) bound HLA-A*0301 or HLA-A*1101 with an affinity ≦100 nM. Twenty-two of these (96%) bound ≧3 alleles with an affinity of ≦500 nM. Eighteen of the 23 (78%) were cross-reactive when the more stringent cut-off of 200 nM was applied.

Additionally, six peptides were tested for immunogenicity in 3-4 donors. When this data is taken into account for those peptides that bind at ≦100 nM to the primary allele and at ≦200 nM to at least 2 additional supertype alleles, 6/6 (CEA.241.KIO, CEA.61, CEA.61, HBER2/nev.681, HER/nev.754, and p53.172.B5KIO) (1000%) are immunogenic and 4/6 (CEA.61, HER2/nev.681, HER/nev.754, and p53.172.B5KIO) (67%) induce tumor-reactive CTLs. These results are similar to those observed with TAA-derived HLA-A2-specific peptides. Keogh, et al. J Immunol. 167(2):787-96 (2001).

The analog peptides were analyzed in the same way. Thirty-nine analogs with either HLA-A*0301 or A*1101 binding affinity ≦100 nM were identified from Tables 4a-d. All 39 (1000%) were demonstrated to be cross-reactive with at least 2 additional supertype alleles with an affinity ≦500 nM. Applying a binding cut-off of 200 nM for supertype cross-reactivity, 27/39 (69%) of the analog peptides were degenerate binders.

The same analysis was performed with the HLA-B7-restricted peptides (derived from Tables 17a-d) although the sample size was much smaller than that for A3. Five wildtype peptides and 5 analogs were identified to have binding affinities ≦100 nM. All 10 (1000%) were also cross-reactive with at least 2 other alleles of the B7 superfamily. When the binding affinity cut-off for cross-reactivity was set at 200 nM or less, 2 out of 5 (40%) peptides were cross-reactive in the case of both wildtype and analog peptides. While this is lower than the 78% observed for A3, this is likely to be due to the small number of peptides available for analysis.

Selection of HLA-A3, -B7, -A1 and -A24 Candidate Peptides for Immunotherapy

Criteria for Selection of Vaccine Candidates

Desirable criteria for the inclusion of an epitope in an immunotherapeutic cancer vaccine are that the epitope bind to three or more alleles of a given superfamily with an affinity ≦500 nM and that such an epitope induces a specific CTL response recognizing target tumor cells. An analysis of Epimmune's accumulated database and the results from primary immunogenicity screening of A2 supertype cancer antigen-derived epitopes in particular provided several important findings. First, a binding affinity of ≦500 nM is highly predictive of immunogenicity. However, peptides that bind with affinities of 201-500 nM did not induce tumor-reactive CTLs. Additionally, it was demonstrated that when a more strict binding affinity cut-off of ≦200 nM was applied to wildtype peptides, 76% of the TAA peptides induced CTLs that recognize the endogenously processed and presented epitope. In the course of the same set of experiments, analogs which bound with an IC₅₀ of 200 nM or less, and for which primary immunogenicity (recognition of wildtype peptide-pulsed targets) could be demonstrated, were studied. These analogs induced CTLs capable of recognizing tumor cell targets and/or wildtype peptides in at least 75% of the cases. It was concluded from this analysis that binding affinity was highly predictive of immunogenicity and that a binding affinity of 200 nM was indicative of a peptides ability to induce tumor reactive CTLs. Therefore, utilizing a binding cut-off of ≦200 nM to select the HLA-A3 and -B7 peptide candidates described herein increases the likelihood of identifying epitopes and reduces the number of peptides to be screened in vitro.

Currently, supertype binding assays are unavailable for A*0101 and A*2402. However, work done with HCV and HBV peptides by researchers at Epimmune as well as studies performed by Doolan (1997) and Threlked (1997) with malaria and HIV-derived peptides, respectively, demonstrated that peptides with a binding affinity ≦100 nM were often degenerate binders. Therefore, A1 and A24 candidate peptides were selected on the basis of a stringent (≦100 nM) binding affinity to the primary allele of the A1 and A24 superfamilies. These selection criteria should ensure that these peptides are not only likely to be cross-reactive, but also immunogenic.

Another factor we wished to consider in the selection of HLA-A3 and -B7-restricted candidates was to allow for a combination of both wild-type peptides and analogs. Using a wildtype peptide increases the likelihood that the peptide is endogenously processed and presented. However, analogs may play an important role, particularly in the case of tumor antigens, because they could more easily overcome T cell tolerance and allow generation of a more multi-specific response. Furthermore, it would be predicted that a more vigorous CTL response can be induced by analoging at primary anchor positions of a peptide to increase binding affinity. In the case of HLA-A1 and HLA-A24-restricted peptides, wildtype peptides may be relatively preferred over analogs until assays are available to determine cross-reactive binding of the parent wildtype peptide. However, analogs should still be considered to provide the greatest coverage of each allele and tumor antigen.

To summarize, candidate peptides are selected on the basis of a binding affinity ≦200 mM and crossreactive binding ≧3 alleles of the HLA-A3 or -B7 superfamilies, or a binding affinity ≦100 nM for HLA-A1 and -A24 and primary immunogenicity and/or endogenous recognition for analogs.

HLA-A3 Cross-Reactive Vaccine Candidates

Using a binding affinity threshold of 200 nM or less for each allele to define supertype cross-reactivity and considering in vitro immunogenicity data where available, six to eight potential vaccine candidates have been identified for each antigen (Table 20).

CEA: When multiple peptides from a single region met the minimum criteria, the peptide that bound the greatest number of alleles was the preferred candidate. CEA.636, CEA.656, CEA.376, CEA.554, and CEA.420V2 were the most highly cross-reactive of the peptides from each of those regions. Lastly, CEA.61 and CEA-241K10 were selected on the basis of their demonstrated ability to induce CTLs in vitro that recognize endogenous targets (CEA.61) or the wildtype peptide (CEA.241K10). The V2 analog of the CEA.241 peptide is a back-up candidate because it meets the minimum criteria but immunogenicity has not yet been demonstrated. When considered as a group, four to seven of the different CEA-derived peptides bind each of the alleles of the A3 superfamily, providing broad population coverage of this antigen.

HER2/neu: For HER2/neu, 8 peptide candidates were identified from the transmembrane or intracellular domain of the protein. There are six candidates that met the criterion of cross-reactive binding to 3 or more alleles described in the previous section. Five of these are wildtype peptides and one is an analog. HER2/neu.669, HER2/neu.852, HER2/neu.860V2, HER2/neu.889, HER2/neu.972 and HER2/neu.997 bound to 23 alleles with an affinity ≦900 nM and are therefore considered to be vaccine candidates representative of 6 distinct protein regions. HER2/neu.669 and HER2/neu.852 were also capable of inducing a CTL response in normal donors. It should be noted that HER2/neu.889 does not meet the binding criteria for either A*0301 or A*1101 but is included to increase coverage of A*3101, A*3301 and A*6801. Two additional wildtype peptides, HER2/neu.681 and HER2/neu.754, that are cross-reactive with 2 alleles and are also included because of available data demonstrating not only immunogenicity but also tumor target recognition by the CTLs induced (Kawashima, 1999). Three to eight of the candidate peptides within this group bound to each of the 5 alleles.

MAGE2/3: Six candidate peptides were identified for MAGE2/3 (four MAGE2 and two MAGE3), consisting of 3 wildtype and 3 analog peptides. Each allele within the supertype is covered by at least 2 peptides within this group. One of them, the MAGE2.73 peptide has been shown to be immunogenic and induce tumor reactive CTLs.

p53: Motif analysis of the p53 protein, determination of binding affinity, and analoging led to the identification of 6 non-redundant candidates. A somewhat greater fraction of candidates is represented by analogs in this case, probably reflective of the relatively small size of the p53 protein. All six peptides (2 wildtype and 4 analogs) bound the A*0301 allele, five bound A*1101 and A*3101, two bound A*3301 and three bound A*6801. Immunogenicity has already been demonstrated for two of the analogs, p53.101K10 and p53.172B5K10. Additionally it was demonstrated that p53.172B5K10-specific CTLs were able to lyse wildtype-peptide coated targets and p53 transfected tumor targets.

Summarizing, there are seven non-redundant CEA-derived peptides (5 wildtype and 2 analogs), 8 HER2/neu-derived peptides (including one analog), 6 peptides each from MAGE2/3 (3 wild-type and 3 analogs) and p53 which is represented by 2 wild-type peptides and 4 analogs.

HLA-B7 Vaccine Candidates

Using a binding affinity threshold of 200 nM or less for each allele to define supertype cross-reactivity, one to three potential candidate peptides have been identified for each antigen (Table 21). One CEA-derived wildtype peptide binds 4 alleles, one HER2/neu-derived wildtype peptide binds four alleles, and one wildtype p53 peptide binds 3 alleles. One wildtype and 2 analog peptides were identified from MAGE2/3 that bind 3-4 alleles. MAGE3.196110 is also included as a candidate (despite the fact that it did not meet the binding criteria for B*0702) because it provides coverage of B*5101, B*5301 and B*5401. Although, almost all the HLA-B7-restricted peptides have been assayed for binding, this allele remains underrepresented in the number of vaccine candidates identified.

HLA-A1 Vaccine Candidates

The selection of HLA-A*0101 wildtype vaccine candidates was based on a binding affinity ≦100 nM. In the case of analog candidates the same binding criteria was applied and, in addition, the parent (wildtype) peptide had to bind ≦5000 nM.

CEA: When these criteria were applied to the peptides listed in Tables 6a-d, the choice of HLA-A*0101 restricted vaccine candidates was straightforward. They include seven non-overlapping CEA peptides (Table 22). Of these, 4 are wildtype peptides and 3 are analog peptides. CEA.418D3 was selected as the candidate from the group of candidates around that protein position because the affinity of the wildtype peptide was ≦500 nM. In the case of CEA.616, the wildtype peptide is the preferred candidate because the analog provides only a marginal (less than 2-fold) increase in binding affinity.

HER2/neu: Four wildtype candidates and 5 analogs derived from HER2/neu met the criteria for the selection of candidate peptides (Table 22). Wherever possible the wildtype peptide was the preferred candidate (eg. HER2/neu.826.T2, HER2/neu.869, HER2/neu.899 and HER2/neu.1213). In the case of the analog peptides, the 5 chosen were clearly associated with improved binding affinities. HER2/neu.997T2 was preferred over HER/neu.996D3 because the higher affinity of the 9-mer wildtype peptide makes it more likely to be presented by the MHC class I molecules and recognized by HER2/neu.996-specific CTLS.

MAGE2/3: Table 22 also lists the eight A1-restricted selected epitopes (four wildtype candidates and 4 analogs) derived from MAGE2 and MAGE3 that met the criteria for the selection of candidates. Once again, wherever possible the wildtype peptide was the preferred candidate (eg. MAGE2.247, MAGE3.246, MAGE3.68 and MAGE3.168). MAGE3.168 has also been previously demonstrated to be an epitope (Tuting, 1998). In the case of the analog peptides, the four chosen are clearly associated with improved binding affinity (≦100 nM).

p53: Finally, a total of four p53-derived peptides have been selected, each representing a discrete region of the protein (Table 22). p53.117 and p53.226 both have a binding affinity ≦100 nM and were therefore chosen as the wildtype candidate peptides. In the case of p53.98T2 and p53.196D3 an approximately 25 to 50-fold increase in binding affinity over their wildtype counterparts was demonstrated. Correspondingly, these analogs were also selected as candidates.

HLA-A24 Vaccine Candidates

As was described above for HLA-A1 epitopes, the selection of HLA-A*2402 wildtype vaccine candidates was based on binding ≦100 nM and immunogenicity data generated at Epimmune or described in the literature. Analog candidates had to both bind ≦100 nM and be associated with a wildtype peptide that binds ≦5000 nM. When a wildtype peptide and corresponding analog both bound ≦100 nM, the wildtype was the preferred choice.

When these criteria were applied to the peptides listed in Tables 7a-d, the HLA-A*2401 restricted vaccine candidate peptides listed in Table 11 were obtained. These included eleven non-overlapping CEA peptides. Of these, 9 are wildtype peptides and 2 are analog peptides.

Similarly, three wildtype candidates and 1 analog derived from Her2/neu met the criteria for the selection of candidate peptides (Table 23). As stated above, the wildtype peptide was the preferred candidate (eg. HER2/neu.780, HER2/neu.907 and HER2/neu.951 (1-mer)). In the case of the analog selected, HER2/neu.968Y2, binding affinity was significantly improved as compared to the wildtype parent counterpart.

Seven wildtype candidate peptides and 3 analog peptides derived from MAGE2 and MAGE3 were identified (Table 23). It can be noted that, the 3 analog peptides chosen (MAGE2.97Y2, MAGE2.175F10 and MAGE3.175F10) were all associated with binding affinity increased more than approximately 15-fold.

Lastly, two p53-derived peptides have also been selected, each representing a discrete region of the protein (Table 23). p53.102 and p53.125 both, have a binding affinity ≦100 nM.

By applying the criterion of a 100 nM cut-off to identify HLA-A24-restricted peptides, a number of candidates have been identified for each antigen. The CEA candidate list includes 9 wildtype and 2 analog peptides. One analog and 3 wildtype peptides derived from the transmembrane and/or intracellular domain of HER2/neu have been identified. A total of 10 MAGE2/3-derived peptides (7 wildtype and 3 analogs) and 2 wildtype p53-derived peptides were identified.

Discussion:

These studies describe the selection of candidates for a therapeutic vaccine utilizing peptides carrying motifs representative of the A3, B7, A1 and A24 superfamilies. In the A3 and B7 supertypes, candidate selection is based on cross-reactive binding affinity. For HLA-A1 and -A24, selection is based solely on binding to A*0101 and A*2402, respectively.

Analoging strategies for HLA-A3, -B7, -A1 and -A24-restricted peptides were also described. Sidney, et al. (2001) (supra). Briefly, the Epimmune analog strategy was first documented for HLA-A2-restricted peptides. Ruppert, et al. PNAS 90(7):2671-75 (1993), also, Grey, et al. Clin. Exp. Rheumatol. Suppl. 8:S47-50 (1993), and researchers at Epimmune (EPI-45-99) demonstrated that substituting preferred amino acids for sub-optimal residues at primary anchor positions increased binding to the 5 predominant alleles of the A2 superfamily. Enhanced immunogenicity of analogs was demonstrated both in mice (Sarobe, 1998) and patients (Rosenberg, 1998). Tumor cell recognition was also observed, indicating that these peptide modifications do not affect the ability of the analog-specific CTL to recognize the endogenously expressed peptide. For A3 supertype molecules, T or V were determined to be optimal at position 2 and K or R optimal at the C-terminus to utilize as substitution residues with the greatest potential for improved crossbinding capacity. For B7 supertype alleles, proline is required at position 2. Although each molecule of the B7 superfamily prefers a different residue at the C-terminus (L, Y, L W or A), isoleucine is the optimal C-terminal substitution residue to improve B7 superfamily crossbinding. Optimal residues for HLA-A1-restricted peptides are T at position 2, D at position 3, and Y at the C-terminus. Lastly, Y at position 2 and F at the C-terminus are recommended for the A24 allele. These strategies were implemented to identify additional binders to supplement population coverage for the CEA, HER2/neu, MAGE2/3 and p53 antigens.

Using cross-reactive binding affinities ≦200 nM for A3 and B7-restricted peptides, binding affinities ≦100 nM for A1 and A24-restricted peptides, and primary immunogenicity and tumor recognition when that data was available, a total of 88 wildtype and analog peptides were selected as candidates for a therapeutic vaccine (Table 24). There are 6-8 A3-restricted candidates for each of the four tumor antigens. In the B7 supertype arena, only 1-3 candidates were identified for each TAA. This is due in part to the requirement that proline be present at primary anchor position 2, which is severely limiting by the number of tolerated residues alone. Couple this with the fact that B7 peptides bind on average three-fold less well that other supertypes, roughly 10% of motif-positive peptides, the low number of identified TAA-derived B7 candidates is expected. However, including A1 and A24-resticted peptides in a non-A2 vaccine would increase coverage of black, Hispanic, and Asian populations and compensate for the paucity of B7 candidate peptides. Four to nine A1-restricted peptides and two to eleven A24-restricted peptides binding ≦100 nM were identified for each antigen. Overall, there are 26 CEA, 22 Her2/neu, 27 MAGE2/3 and 13 p53 potential candidates.

Analysis of A2 peptides that were able to induce both peptide and tumor reactive CTLs led to the observation that when a binding cut-off of ≦200 nM and supertype crossreactivity 23 alleles was employed, 76% of the wildtype peptides and 50% of the analogs induced tumor reactive CTLs. When primary immunogenicity data (wildtype recognition) was included, 81% of wildtype peptides and 75% of analogs were identified as tumor epitopes. When a 200 nM cut-off was used, to define cross-reactivity to the A3 superfamily alleles, 78% of wildtype peptides and 69% of analogs that bound A*0301 or A*1101 with an IC₅₀ ≦100 nM were degenerate binders. A subset of A3, B7, A1 and A24 peptides described herein were tested for immunogenicity and tumor cell reactivity. When binding and cross-reactivity, as previously described for A2 peptides, were considered, 12/13 (92%) induce CTLs that recognize the wildtype peptide and 8/11 (72%) induce CTLs that recognize the endogenously processed peptide confirming the correlation between binding affinity and immunogenicity.

Assays have been established and validated to measure the capacity of a peptide to bind the 5 most predominant alleles of the A3 and B7 superfamilies, and subsequently select the cross-reactive (degenerate) candidate peptides. Supertypes for A1 and A24 have been proposed, but binding assays are currently available only for the primary alleles of those superfamilies, A*0101 and A*2402. Analysis of infectious disease-derived peptides indicates that binding affinity 5100 nM is predictive of degenerate binding (≦500 nM) to at least 2 other alleles of the superfamily. The same analysis of the A3 and B7 TAA-derived peptides described in this example validates this finding. Of 28 wildtype peptides with a binding affinity ≦100 nM for A*0301/A*1101 or B*0702, 27 (96%) bound 2 or more additional alleles with an affinity ≦500 nM. This finding indicates that TAA-derived peptides behave in a similar fashion to infectious disease-derived peptides and that a binding affinity of 100 nM or less for the primary allele of the superfamily is highly predictive of cross-reactivity.

In conclusion, this example describes the identification of peptide candidates derived from 4 TAA and covering multiple alleles. These candidates include both wildtype peptides and fixed anchor analogs which are either immunogenic or have a binding affinity predicted to be conducive to immunogenicity.

It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, patent applications and sequence listings cited herein are hereby incorporated by reference in their entirety for all purposes. TABLE 1 Overview of current cancer vaccine approaches. APPROACH DESCRIPTION ISSUES STRENGTHS Whole Cell Involve the administration of Often difficult to Likely to have Vaccines whole cancer cells with obtain tumor cells novel TAA adjuvants which serve to Patient variability potentiate the immune response Single patient product Has relatively low concentration of relevant TAA epitopes Cell Lysate Consist of lysed allogeneic Often difficult to Likely to have Vaccines cancer cell membrane particles obtain tumor cells novel TAA that are ingested by macrophages Patient variability and presented as tumor antigens Single patient product to effector cells Has relatively low concentration of relevant TAA epitopes Idiotypic Contain proteins derived from Often difficult to Specific TAA Vaccines individual patient tumors or from obtain tumor cells specific tumor types Patient variability Single patient product Has relatively low concentration of relevant TAA epitopes Whole Limited disease Complex Antigen coverage “natural” Vaccines Difficult to break immune tolerance responses may be elicited Relatively easy single compound manufacture Viral Consist of vaccinia virus Often difficult to oncolysate infected cancer cell, lysed to obtain tumor cells vaccines form membrane segments Not always possible expressing both vaccinia and to infect cancer cells cancer cell antigens Patient specific treatment Has relatively low concentration of relevant TAA epitopes Shed antigen Similar to whole cell and lysate Difficult to purify Likely to have vaccines vaccines but are partially antigens novel TAA purified Patient specific treatment Has relatively low concentration of relevant TAA epitopes Genetically A number of avenues are being Very difficult to Cells contain modified explored including the obtain tumor tissues novel TAA tumor cell transduction of cells with GM- and grow to allow and adjuvants vaccines CSF stable transduction Patient specific treatment Peptide Synthetic peptides are produced Need to choose Single Vaccines that correspond to tumor correct peptides to preparation associated antigens. Designed to elicit an effective used for stimulate a cytotoxic T-Cell immune response multiple response (CTL) Restriction to HLA patients and subtype or HLA possibly supertypes multiple diseases Possible to combine various antigens/ targets Reproducible antigen production Able to break tolerance Able to elicit responses to subdominant epitopes Can be directed to supertypes for broad population coverage Carbohydrate Synthetically produced tumor May need CTL Single vaccines associated carbohydrates, response as well as preparation designed to stimulate an humoral response used for antibody response against the Carbohydrate multiple carbohydrate antigens antigens are HTL patients and dependent possibly multiple diseases

TABLE 2 POSITION POSITION POSITION C Terminus (Primary 2 (Primary Anchor) 3 (Primary Anchor) Anchor) SUPERMOTIFS A1 T, I, L, V, M, S F, W, Y A2 L, I, V, M, A, T, Q I, V, M, A, T, L A3 V, S, M, A, T, L, I R, K A24 Y, F, W, I, V, L, M, T F, I, Y, W, L, M B7 P V, I, L, F, M, W, Y, A B27 R, H, K F, Y, L, W, M, I, V, A B44 E, D F, W, L, I, M, V, A B58 A, T, S F, W, Y, L, I, V, M, A B62 Q, L, I, V, M, P F, W, Y, M, I, V, L, A MOTIFS A1 T, S, M Y A1 D, E, A, S Y A2.1 L, M, V, Q, I, A, T V, L, I, M, A, T A3 L, M, V, I, S, A, T, F, K, Y, R, H, F, A C, G, D A11 V, T, M, L, I, S, A, K, R, Y, H G, N, C, D, F A24 Y, F, W, M F, L, I, W A*3101 M, V, T, A, L, I, S R, K A*3301 M, V, A, L, F, I, S, T R, K A*6801 A, V, T, M, S, L, I R, K B*0702 P L, M, F, W, Y, A, I, V B*3501 P L, M, F, W, Y, I, V, A B51 P L, I, V, F, W, Y, A, M B*5301 P I, M, F, W, Y, A, L, V B*5401 P A, T, I, V, L, M, F, W, Y Bolded residues are preferred, italicized residues are less preferred: A peptide is considered motif-bearing if it has primary anchors at each primary anchor position for a motif or supermotif as specified in the above table.

TABLE 2a POSITION POSITION POSITION C Terminus (Primary 2 (Primary Anchor) 3 (Primary Anchor) Anchor) SUPERMOTIFS A1 T, I, L, V, M, S F, W, Y A2 V, Q, A, T I, V, L, M, A, T A3 V, S, M, A, T, L, I R, K A24 Y, F, W, I, V, L, M, T F, I, Y, W, L, M B7 P V, I, L, F, M, W, Y, A B27 R, H, K F, Y, L, W, M, I, V, A B58 A, T, S F, W, Y, L, I, V, M, A B62 Q, L, I, V, M, P F, W, Y, M, I, V, L, A MOTIFS A1 T, S, M Y A1 D, E, A, S Y A2.1 V, Q, A, T* V, L, I, M, A, T A3.2 L, M, V, I, S, A, T, F, K, Y, R, H, F, A C, G, D A11 V, T, M, L, I, S, A, K, R, H, Y G, N, C, D, F A24 Y, F, W F, L, I, W *If 2 is V, or Q, the C-term is not L Bolded residues are preferred, italicized residues are less preferred: A peptide is considered motif-bearing if it has primary anchors at each primary anchor position for a motif or supermotif as specified in the above table.

TABLE 3 POSITION C-

terminus SUPER- MOTIFS A1 1° Anchor 1° T, I, L, Anchor V, M, S F, W, Y A2 1° Anchor 1° L, I, V, Anchor M, A L, I, T, Q V, M, A, T A3 pre- 1° Anchor Y, F, Y, F, Y, F, P, 1° ferred {overscore (V, S, M, A,)} W, W, W, (4/5) Anchor T, L, I (4/5) (3/5) (4/5) R, K delete- D, E (3/5); D, E, rious P, (5/5) (4/5) A24 1° Anchor 1° Y, F, W, Anchor I, V, F, I, L, M, T Y, W, L, M B7 pre- F, W, 1° Anchor F, W, F, W, 1° ferred Y (5/5) P Y Y, Anchor L, I, V, (4/5) (3/5) V, I, M, (3/5) L, F, M, W, Y, A delete- D, E (3/5); D, E, G, Q, N, D, E, rious P(5/5); (3/5) (4/5) (4/5) (4/5) G(4/5); A(3/5); Q, N, (3/5) B27 1° Anchor 1° R, H, K Anchor F, Y, L, W, M, V, A B44 1° Anchor 1° E, D Anchor F, W, Y, L, I, M, V, A B58 1° Anchor 1° A, T, S Anchor F, W, Y, L, I, V, M, A B62 1° Anchor 1° Q, L, I, Anchor V, M, P F, W, Y, M, I, V, L, A MOTIFS A1 pre- G, F, 1° Anchor D, E, Y, F, P, D, E, Y, F, 1° 9-mer ferred Y, W, S, T, M, A, W, Q, N, W, Anchor Y delete- D, E, R, H, A, G, A, rious K, L, I, V M, P, A1 pre- G, R, A, S, T, 1° G, S, A, S, L, I, D, E, 1° 9-mer ferred H, K C, L, I Anchor T, C, T, C, V, M, Anchor V, M, D, E, Y A, S delete- A R, H, K, D, E, P, Q, R, H, P, G, G, P, rious D, E, N, K, P, Y, F, W, POSITION

or C-ter- C-ter-

minus minus A1 pe- Y, F, W, 1° Anchor D, E, A, A, Y, F, W, P, A, S, G, D, E, P, 1° 10-mer ferred S, T, M Q, N, Q, N, T, C, Anchor Y delete- G, P, R, H, K, D, E, R, H, K, Q, N, A R, H, K, R, H, K, A rious G, L, I Y, F, W, V, M, A1 pre- Y, F, W, S, T, C, 1° Anchor A, Y, F, W, P, G, G, Y, F, 1° 10-mer ferred L, I, V D, E, A, S W, Anchor M, Y delete- R, H, K, R, H, K, P, G, P, R, Q, N, rious D, E, H, K, P, Y, F, W, A2.1 pre- Y, F, W, 1° Anchor Y, F, W, S, T, C, Y, F, W, A, P 1° 9-mer ferred L, M, I, Anchor V, Q, V, L, A, T I, M, A, T delete- D, E, P, D, E, R, R, K, H D, E, R, rious K, H K, H A2.1 pre- A, Y, F, 1° Anchor L, V, I, M, G, G, F, Y, 1° 10-mer ferred W, L, M, I, W, L, Anchor V, Q, V, I, M, V, L, A, T I, M, A, T delete- D, E, P, D, E, R, K, H, P, R, K, H, D, E, R, K, rious A, R, K, H, H, A3 pre- R, H, K, 1° Anchor Y, F, W, P, R, H, A, Y, F, W, P, 1° ferred L, M, V, K, Y, Anchor I, S, F, W, K, Y, A, T, F, R, H, C, G D F, A delete- D, E, P, D, E rious A11 pre- A, 1° Anchor Y, F, W, Y, FW, A, Y, F, W, Y, FW, P, 1° ferred V, T, L, Anchor M, I, S, K, , A, G, N, RY, H C, D, F delete- D, E, P, A G, rious A24 pre- Y, F, W, 1° Anchor S, T, C Y, F, W, Y, F, W, 1° 9-mer ferred R, H, K, {overscore (Y, F, W, M)} Anchor F, L, I, W delete- D, E, G, D, E, G, Q, N, P, D, E, R, G, A, Q, N, rious H, K, A24 pre- 1° Anchor P, Y, F, P, 1° 10-mer ferred {overscore (Y, F, W, M)} W, P, Anchor F, L, I, W delete- G, D, E Q, N R, H, K D, E A Q, N, D, E, rious A, A3101 pre- R, H, K, 1° Anchor Y, F, W, P, Y, F, W, Y, F, W, A, P, 1° ferred M, V, T, Anchor A, L, I, S R, K delete- D, E, P, D, E, A, D, E, D, E, D, E, D, E, rious A3301 pre- 1° Anchor Y, F, W A, Y, F, W 1° ferred M, V, A, Anchor L, F, R, K I, S, T delete- G, P D, E rious A6801 pre- Y, F, W, 1° Anchor Y, F, W, Y, F, W, P, 1° ferred S, T, C, {overscore (A, V, T, M,)} L, I, Anchor S, L, I V, M R, K delete- G, P, D, E, G, R, H, K, A, rious B0702 pre- R, H, K, 1° Anchor R, H, K, R, H, K, R, H, K, R, H, K, P, A, 1° ferred F, W, Y, P Anchor L, M, F, W, Y, A, I, V delete- D, E, Q, D, E, P, D, E, D, E, G, D, E, Q, N, D, E, rious N, P, B3501 pre- F, W, Y, 1° Anchor F, W, Y, F, W, Y, 1° ferred L, I, V, P Anchor M, L, M, F, W, Y, I, V, A delete- A, G, P, G, G, rious B51 pre- L, I, V, 1° Anchor F, W, Y, S, T, C, F, W, Y, G, F, W, Y, 1° ferred M, F, P Anchor W, Y, L, I, V, F, W, Y, A, M delete- A, G, P, D, E, G, D, E, Q, N, G, D, E, rious D, E, R, H, K, S, T, C, B5301 pre- L, I, V, 1° Anchor F, W, Y, S, T, C, F, W, Y, L, I, V, F, W, Y, 1° ferred M, F, P M, F, Anchor W, Y, W, Y, I, M, F, W, Y, A, L, V delete- A, G, P, G, R, H, K, D, E, rious Q, N, Q, N, B5401 pre- F, W, Y, 1° Anchor F, W, Y, L, I, A, L, I, F, W, Y, 1° ferred P L, I, V, V, M, V, M, A, P, Anchor M, A, T, I, V, L, M, F, W, Y delete- G, P, Q, G, D, E, R, H, K, D, E, Q, N, D, D, E, rious N, D, E, S, T, C, D, E, G, E, Italicized residues indicate less preferred or “tolerated” residues. The information in Table II is specific for 9-mers unless otherwise specified. Secondary anchor specificities are designated for each position independently.

TABLE 4 POSITION MOTIFS

DR4 preferred F, M, Y, M, T, I, V, S, T, M, H, M, H L, I, C, P, A, V, W, L, I, M, deleterious W, R, W, D, E DR1 preferred M, F, L, P, A, M, Q, V, M, A, M, A, V, M I, V, T, S, P, W, Y L, I, C, deleterious C, C, H F, D, C, W, D, G, D, E, D DR7 preferred M, F, L, M, W, A, I, V, M, M, I, V I, V, S, A, C, W, Y, T, P, L, deleterious C, G, G, R, D, N, G DR M, F, L, V, M, S, Supermotif I, V, T, A, C, W, Y P, L, I DR3 MOTIFS

motif a L, I, V, preferred M, F, Y D motif b L, I, V, D, N, Q, E, preferred M, F, A, Y S, T K, R, H Italicized residues indicate less preferred or “tolerated” residues.

TABLE 5 HLA- Allelle-specific HLA-supertype members supertype Verified^(a) Predicted^(b) A1 A*0101, A*2501, A*2601, A*0102, A*2604, A*3601, A*2602, A*3201, A*2902 A*4301, A*8001 A2 A*0201, A*0202, A*0203, A*0208, A*0210, A*0211, A*0204, A*0205, A*0206, A*0212, A*0213 A*0207, A*0209, A*0214, A*6802, A*6901 A3 A*0301, A*1101, A*3101, A*0302, A*1102, A*2603, A*3301, A*6801 A*3302, A*3303, A*3401, A*3402, A*6601, A*6602, A*7401 A24 A*2301, A*2402, A*3001 A*2403, A*2404, A*3002, A*3003 B7 B*0702, B*0703, B*0704, B*1511, B*4201, B*5901 B*0705, B*1508, B*3501, B*3502, B*3503, B*3503, B*3504, B*3505, B*3506, B*3507, B*3508, B*5101, B*5102, B*5103, B*5104, B*5105, B*5301, B*5401, B*5501, B*5502, B*5601, B*5602, B*6701, B*7801 B27 B*1401, B*1402, B*1509, B*2701, B*2707, B*2708, B*2702, B*2703, B*2704, B*3802, B*3903, B*3904, B*2705, B*2706, B*3801, B*3905, B*4801, B*4802, B*3901, B*3902, B*7301 B*1510, B*1518, B*1503 B44 B*1801, B*1802, B*3701, B*4101, B*4501, B*4701, B*4402, B*4403, B*4404, B*4901, B*5001 B*4001, B*4002, B*4006 B58 B*5701, B*5702, B*5801, B*5802, B*1516, B*1517 B62 B*1501, B*1502, B*1513, B*1301, B*1302, B*1504, B*5201 B*1505, B*1506, B*1507, B*1515, B*1520, B*1521, B*1512, B*1514, B*1510 ^(a)Verified alleles include alleles whose specificity has been determined by pool sequencing analysis, peptide binding assays, or by analysis of the sequences of CTL epitopes. ^(b)Predicted alleles are alleles whose specificity is predicted on the basis of B and F pocket structure to overlap with the supertype specificity.

TABLE 6 Identified CTL Epitopes for an A2 Vaccine CTL¹ HLA-A2 Binding No. A2 Wild- Sequence Affinity (IC50 nM) Alleles type Source (SEQ ID NO:) A*0201 A*0202 A*0203 A*0206 A*6802 Bound Sequence Sequence⁴ Tumor CEA.24V9 LLTFWNPPV (19) 16 307 26 56 952 4 1/1 TBD² 1/1 CEA.233V10 VLYGPDAPTV (1) 26 430 16 206 952 4 3/4 2/2 1/4 CEA.605V9 YLSGANLNV (2) 73 13 13 80 1600 4 4/4 3/4 1/4 CEA.687 ATVGIMIGV (3) 36 8.8 20 11 0.80 5 1/1 1/1 1/1 CEA.691 IMIGVLVGV (16) 69 62 13 106 89 5 8/8 8/8 4/7 p53.25V11 LLPENNVLSPV (4) 38 4 4 9 30 5 2/3 1/3 1/3 p53.139L2 KLCPVQLWV (5) 122 239 29 23 —³ 4 2/5 2/3 1/3 p53.139L2B3 KLBPVQLWV (6) 34 8.7 20 11 — 4 3/4 2/3 1/2 p53.149L2 SLPPPGTRV (7) 122 226 13 9250 140 4 2/3 1/3 0/3 p53.149M2 SMPPPGTRV (8) 172 215 13 425 667 4 2/4 2/4 2/4 Her2/neu.5 ALCRWGLLL (12) 100 —² 278 — — 2 2/2 2/2 2/2 Her2/neu.48 HLYQGCQVV (14) 139 307 13 514 1143 3 3/4 3/4 1/3 Her2/neu.369 KIFGSLAFL (17) 36 9 19 23 3333 4 10/11 10/11  7/11 Her2/neu. KLFGSLAFV (9) 5.8 7.5 19 17 1270 4 4/4 3/4 2/4 369L2V9 Her2/neu. KVFGSLAFV (10) 20 19 769 15 29 4 4/4 3/4 2/4 369V2V9 Her2/neu.435 ILHNGAYSL (15) 75 358 100 569 — 3 5/5 5/5 3/5 Her2/neu.665 VVLGVVFGI (23) 14 — 2500 430 2000 2 4/8 4/8 1/1 Her2/neu.689 RLLQETELV (22) 21 — 625 34 — 2 4/8 4/8 1/1 Her2/neu.773 VMAGVGSPYV (11) 200 391 13 3700 — 3 2/4 2/4 1/4 Her2/neu.952 YMIMVKCWMI 20 307 83 116 267 5 2/3 2/3 2/3 (25) MAGE2.157 YLQLVFGIEV (24) 50 165 345 370 9302 4 3/3 3/3 1/3 MAGE3.159 QLVFGIELMEV 7.9 74 217 185 267 5 3/3 3/3 1/3 (21) MAGE3.112 KVAELVHFL (18) 69 29 14 168 17 5 3/4 3/4 3/4 MAGE3.160 LVFGIELMEV (20) 29 20 7.7 28 14 5 4/4 4/4 1/4 MAGE3.271 FLWGPRALV (13) 31 43 14 336 40 5 4/4 4/4 2/4 ¹Number of donors yielding a positive response/total tested. ²To be determined ³— indicates binding affinity ≦10,000 nM. ⁴For peptides that are not analogs, “Sequence” and “Wild-type Sequence” provide the same information

TABLE 7 Expression of Tumor Associated Antigen (TAA) % of Tumors Expressing the TAA TAA Colon Cancer Breast Cancer Lung Cancer CEA 95 50 70 P53 50 50 40-60 MAGE 2/3 20-30 20-30 35 HER2/neu 28-50 30-50 20-30 Total 99 86-91 91-95

TABLE 8 Incidence and survival rate of patients with breast, colon, or lung Ucancer in the United States Estimated New Cases Estimate 5-Year relative survival rates 1998 Deaths 1998 1974-76 1980-82 1986-1993 Breast 180,300 43,900 75% 77% 80% Colon  95,600 47,700 50% 56% 63% Lung 171,500 160,100 12% 14% 14% Source: Cancer Statistics 1998. January/February 1998, Vol. 48, No. 1

TABLE 9 Summary of CTL Epitopes for an A2 Vaccine CTL No. A2 Recognition A*0201 A*0202 A*0203 A*0206 A*6802 Members Native Sequence ID₅₀ ID₅₀ ID₅₀ ID₅₀ ID₅₀ Cross- Pulsed Tumor Epitope¹ (SEQ ID NO.) (nM)² (nM)² (nM)² (nM)² (nM)² bound Cells Cell CEA.605V9 YLSGANLNV (2) 73³ 13 13 80 1600 4 + + CEA.691 IMIGVLVGV (16) 69 62 13 106 89 5 + + p53.139L2B3 KLBPVQLWV (6) 34 8.7 20 11 —⁴ 4 + + p53.149M2 SMPPPGTRV (8) 172 215 13 425 667 4 + + MAGE3.112 KVAELVHFL (18) 69 29 14 168 17 5 + + MAGE2.157 YLQLVFGIEV (24) 50 165 345 370 9302 4 + + HER2/neu.689 RLLQETELV (22) 21 — 625 34 — 2 + + HER2/neu.665 VVLGVVFGI (23) 14 — 2500 430 2000 2 N.D. + ¹The peptide designations are derived from the target antigen (e.g. CEA) and the numeral relates to the first amino acid in the protein (e.g. 691). Analogs are noted by the amino acid inserted by substitution and the peptide position substituted (e.g. V9). ²HLA binding was measured by a competitive binding assay where lower values indicate greater binding affinity. ³Standard errors corresponding to HLA binding were presented in previous figures. ⁴— indicates binding affinity >10,000 nM.

TABLE 10 Identified CTL Epitopes for an A2 Vaccine CTL¹ HLA-A2 Binding No. A2 Wild- Sequence Affinity (IC50 nM) Alleles type Source (SEQ ID NO:) A*0201 A*0202 A*0203 A*0206 A*6802 Bound Sequence Sequence⁴ Tumor CEA.24V9 LLTFWNPPV (19) 16 307 26 56 952 4 1/1 TBD² 1/1 CEA.233V10 VLYGPDAPTV (1) 26 430 16 206 952 4 3/4 2/2 1/4 CEA.687 ATVGIMIGV (3) 36 8.8 20 11 0.80 5 1/1 1/1 1/1 P53.25V11 LLPENNVLSPV (4) 38 4 4 9 30 5 2/3 1/3 1/3 P53.139L2 KLCPVQLWV (5) 122 239 29 23 —³ 4 2/5 2/3 1/3 Her2/neu.369 KIFGSLAFL (17) 36 9 19 23 3333 4 10/11 10/11  7/11 Her2/neu. KVFGSLAFV (10) 20 19 769 15 29 4 4/4 3/4 2/4 369V2V9 Her2/neu.952 YMIMVKCWMI (25) 20 307 83 116 267 5 2/3 2/3 2/3 MAGE3.159 QLVFGIELMEV (21) 7.9 74 217 185 267 5 3/3 3/3 1/3 MAGE3.160 LVFGIELMEV (20) 29 20 7.7 28 14 5 4/4 4/4 1/4 ¹Number of donors yielding a positive response/total tested. ²To be determined ³— indicates binding affinity ≦10,000 nM. ⁴For peptides that are not analogs, “Sequence” and “Wild-type Sequence” provide the same information

TABLE 11 Population coverage by HLA class I supertype epitopes. Minimal Allelic Frequency Representative HLA Supertype Molecules* Caucasian Black Japanese Chinese Hispanic Average A2 2.1, 2.2, 2.3, 2.5, 45.8 39.0 42.4 45.9 43.0 43.2 2.6, 2.7, 68.02 A3 3, 11, 31, 33, 37.5 42.1 45.8 52.7 43.1 44.2 68.01 B7 7, 51, 53, 35, 54 43.2 55.1 57.1 43.0 49.3 49.5 Total Population Coverage 84.3 86.8 89.5 89.8 86.8 87.4 For A2, all A2 subtypes were included; for A3, the five listed allotypes were used; for B7, several additional allotypes were included based on binding pocket analysis.

TABLE 12 Tumor Associated Antigens and Genes (TAA) ANTIGEN REFERENCE MAGE 1 (Traversari C., Boon T, J. Ex. Med 176: 1453, 1992) MAGE 2 (De Smet C., Boon T, Immunogenetics, 39(2)121-9, 1994) MAGE 3 (Gaugler B., Boon T, J. Ex. Med 179: 921, 1994) MAGE-11 (Jurk M., Winnacker L, Int.J. Cancer 75, 762-766, 1998) MAGE-A10 (Huang L., Van Pel A, J. Immunology, 162: 6849-6854) BAGE (Boel P., Bruggen V, Immunity 2: 167, 1995) GAGE (Eynde V., Boon T, J. Exp. Med 182: 689, 1995) RAGE (Gaugler B., Eynde V, Immunogenetics, 44: 325, 1996) MAGE-C1 (Lucas S., Boon T, Cancer Research, 58, 743-752, 1998) LAGE-1 (Lethel B., Boon T, Int J cancer, 10; 76(6) 903-908 CAG-3 (Wang R-Rosenberg S, J. Immunology, 161: 3591-3596, 1998) DAM (Fleischhauer K., Traversari C, Cancer Research, 58, 14, 2969, 1998) MUC1 (Karanikas V., McKenzie IF, J. clnical investigation, 100: 11, 1-10, 1997) MUC2 (Bohm C., Hanski, Int.J. Cancer 75, 688-693, 1998) MUC18 (Putz E., Pantel K, Cancer Res 59(1): 241-248, 1999) NY-ES0-1 (Chen Y., Old LJ PNAS, 94, 1914-18, 1997) MUM-1 (Coulie P., Boon T, PNAS 92: 7976, 1995) CDK4 (Wolfel T., Beach D, Science 269: 1281, 1995) BRCA2 (Wooster R-Stratton M, Nature, 378, 789-791, 1995) NY-LU-1 (Gure A., Chen, Cancer Research, 58, 1034-41, 1998) NY-LU-7 (Gure A., Chen, Cancer Research, 58, 1034-41, 1998) NY-LU-12 (Gure A., Chen, Cancer Research, 58, 1034-41, 1998) CASP8 (Mandruzzato S., Bruggen P, J. Ex.Med 186, 5, 785-793, 1997) RAS (Sidransky D., Vogelstein B, Science, 256: 102) KIAA0205 (Gueguen M., Eynde, J. Immunology, 160: 6188-94, 1998) SCCs (Molina R., Ballesta AM, Tumor Biol, 17(2): 81-9, 1996) p53 (Hollstein M., Harris CC, Science, 253, 49-53, 1991) p73 (Kaghad M., Caput D, Cell; 90(4): 809-19, 1997) CEA (Muraro R., Schlom J, Cancer Research, 45: 5769-55780, 1985) Her 2/neu (Disis M., Cheever M, Cancer Res 54: 1071, 1994) Melan-A (Coulie P., Boon T, J. Ex.Med, 180: 35, 1994) gp100 (Bakker A., Figdor, J. Ex.Med 179: 1005, 1994) Tyrosinase (Wolfel T., Boon T, E.J. I 24: 759, 1994) TRP2 (Wang R., Rosenberg S. A, J. Ex.Med 184: 2207, 1996) gp75/TRP1 (Wang R., Rosenberg S. A, J. Ex.Med 183: 1131, 1996) PSM (Pinto J. T., Heston W. D. W., Clin Cancer Res 2(9); 1445-1451, 1996) PSA (Correale P., Tsang K, J. Natl cancer institute, 89: 293-300, 1997) PT1-1 (Sun Y., Fisher PB, Cancer Research, 57(1): 18-23, 1997) B-catenin (Robbins P., Rosenberg SA, J. Ex. Med 183: 1185, 1996) PRAME (Neumann E., Seliger B, Cancer Research, 58, 4090-4095, 1998) Telomearse (Kishimoto K., Okamoto E, J Surg Oncol, 69(3): 119-124, 1998) FAK (Kornberg LJ, Head Neck, 20(8): 745-52, 1998) Tn antigen (Wang Bl, J Submicrosc Cytol Path, 30(4): 503-509, 1998) cyclin D1 protein (Linggui K., Yaowu Z, Cancer Lett 130(1-2), 93-101, 1998) NOEY2 (Yu Y., Bat RC, PNAS, 96(1): 214-219, 1999) EGF-R (Biesterfeld S.- Cancer Weekly, Feb15, 1999) SART-1 (Matsumoto H., Itoh K, Japanese Journal of Cancer Research, 59, iss12, 1292-1295, 1998) CAPB (Cancer Weekly, March 29, 4-5, 1999) HPVE7 (Rosenberg S. A. Immunity, 10, 282-287, 1999) p15 (Rosenberg S. A., Immunity, 10, 282-287, 1999) Folate receptor (Gruner B. A., Weitman S. D., Investigational New Drugs, Vol16, iss3, 205-219, 1998) CDC27 (Wang R. F., Rosenberg SA, Science, vol 284, 1351-1354, 1999) PAGE-1 (Chen, J. Biol. Chem: 273: 17618-17625, 1998) PAGE-4 (Brinkmann: PNAS, 95: 10757, 1998) Kallikrein 2 (Darson: Urology, 49: 857-862, 1997) PSCA (Reiter R., PNAS, 95: 1735-1740, 1998) DD3 (Bussemakers M. J. G, European Urology, 35: 408-412, 1999) RBP-1 (Takahashi T., British Journal of Cancer, 81(2): 342-349, 1999) RU2 (Eybde V. D., J. Exp.Med, 190 (12): 1793-1799, 1999) Folate binding (Kim D., Anticancer Research, 19: 2907-2916, 1999) protein EGP-2 (Heidenreich R., Human Gene Therapy, 11: 9-19, 2000)

TABLE 13a Phenotypic frequencies by supertype family. Minimal Population Coverage North American Chi- His- Allelle Caucasian Black Japanese nese panic Average A2 45.8 39.0 42.4 45.9 43.0 43.2 supertype A3 37.5 42.1 45.8 52.7 43.1 44.2 supertype B7 38.6 52.7 48.8 35.5 47.1 44.7 supertype A1 47.1 16.1 21.8 14.7 26.3 25.2 supertype A24 23.9 38.9 58.6 40.1 38.3 40.0 supertype Total 99.0 97.3 100.0 99.1 98.8 98.8

TABLE 13b Phenotypic frequencies by motif type in current study. Minimal Population Coverage North American Chi- His- Allelle Caucasian Black Japanese nese panic Average A2 supertype 45.8 39.0 42.4 45.9 43.0 43.2 A3 supertype 37.5 42.1 45.8 52.7 43.1 44.2 B7 supertype 38.6 52.7 48.8 35.5 47.1 44.7 A1 28.6 10.1 1.4 9.2 10.1 11.9 A24 16.8 8.8 58.1 32.9 26.7 28.7 Total 95.1 90.6 99.1 97.5 94.8 95.4

TABLE 14 Sequence motifs associated with binding-MHC specificity analyzed in the current study Anchor position Allelle p2 p3 C terminus A3 supertype LMIVAST — KR B7 supertype P — LMIVFWYA A1 TSM or DEAS YA A24 YFWM — FLIW — indicates any naturally occurring residue accepted.

TABLE 15 Motif-positive peptides derived from target TAA A3 supertype B7 supertype A1 A24 Antigen Wildtype Analog¹ Wildtype Analog Wildtype Analog Wildtype Analog Total CEA Motif-positive 60 113 79 205 50 81 46 61 695 Tested 52 12 79 92 48 17 15 13 358 Her2/neu Motif-positive 107 194 85 237 32 55 43 69 822 Tested 53 11 85 128 26 55 41 6 364 Mage2/3 Motif-positive 63 113 61 166 27 49 51 81 611 Tested 63 17 61 85 27 13 46 8 321 p53 Motif-positive 78 147 64 188 15 24 20 30 566 Tested 78 24 62 103 14 5 12 2 300 Total Motif-positive 308 567 289 796 124 209 160 241 2694 Tested 247 64 287 408 115 49 144 29 1343 ¹Numbers refer to maximum analogs possible substituting at the p2 or C-terminus anchor position. Substitution at both anchors concurrently not considered. For the B7 supertype, analog numbers presented also include F1 substitutions, a non-anchor substitution.

TABLE 16a CEA-derived A3 supertype crossbinders Number of CTI¹ inductions yielding positive responses/ total tested SEQ Number of Wildtype-pulsed ID HLA Binding (IC₅₀ nM) A3 alleles EHM target Tumor target² NO Sequence Source A*0301 A*1101 A*3101 A*3301 A*6801 crossbound cell line cell line 42 HLFGYSWYK CEA.61 2.2 2.4 20 18 3.5 5 3/4 2/4 43 TISPLNTSYR CEA.241 1594 158 207 569 4.4 3 44 TISPLNTSYK CEA.241.K10 61 182 —³ — 116 3 1/1 0/1 45 TVSPLNTSYR CEA.241.V2 458 55 188 558 8.7 4 46 RTLTLLSVTR CEA.376 524 55 6.0 1036 160 3 47 RVLTLLSVTR CEA.376.V2 344 222 11 6042 667 3 48 PTISFSYTYYR CEA.418 — 46 44 784 57 3 49 TISPSYTYYR CEA.419 3438 21 72 171 3.1 4 50 ISPSYTYYR CEA.420 1342 143 21 518 11 3 51 IVPSYTYYR CEA.420.V2 92 13 26 58 2.6 5 52 RTLTLFNVTR CEA.554 111 13 5.0 1611 99 4 1/1 nt 53 RVLTLFNVTR CEA.554.V2 297 94 9.0 7632 42 4 54 HTQVLFLAK CEA.636 1183 35 106 132 160 4 55 FVSNLATGR CEA.656 5790 122 333 104 8.2 4 ¹A positive response is defined as 10% specific lysis above background in the ⁵¹ Cr release assay or twice background and at least 50 pg IFNγ/well above background in at least one of 48 wells tested. ²The tumor target cell lines used are SW480 (A3−, CEA+) and SW403 (A3+, CEA+). Only tumor target data from wells positive for recognition of peptide-pulsed target cells is shown. ³— indicates binding affinity = 10,000 nM.

TABLE 16b Her2/neu-derived A3 supertype crossbinders Number of CTI¹ inductions yielding positive responses/ total tested Wildtype- SEQ Number of pulsed ID HLA Binding (IC₅₀ nM) A3 alleles EHM target Tumor target² NO Sequence Source A*0301 A*1101 A*3101 A*3301 A*6801 crossbound cell line cell line 56 GVFGILIKR Her2/neu.668 611 182 305 2071 19 3 57 VVFGILIKR Her2/neu.669 100 8.3 13 78 4.0 5 1/3 0/1 58 VVFGILIKRR Her2/neu.669 3667 375 290 193 15 4 59 VVFGILIKKC Her2/neu.669.K9 22 19 3750 —³ 35 3 60 KIRKYTMRR Her2/neu.681 15 3333 16 4028 — 2 3/3 2/3 61 KIRKYTMRK Her2/neu.681.K9 177 1091 6429 — — 1 62 VLRENTSPK Her2/neu.754 28 462 129 290 — 4 4/7 3/6 63 VVRENTSPR Her2/neu.754.V2R9 200 5455 375 126 178 4 64 LLDHVRENR Her2/neu.806 297 — 500 326 5714 3 65 LAARNVLVK Her2/neu.846 190 211 — — 500 3 66 LVARNVLVK Her2/neu.846.V2 42 214 9000 — 205 3 67 LVKSPNHVK Her2/neu.852 23 86 182 784 73 4 0/3 0/1 68 KITDFGLAR Her2/neu.860 65 25 100 — 1633 3 0/1 nt 69 KVTDFGLAR Her2/neu.860.V2 201 76 106 — 133 4 70 MALESILRR Her2/neu.889 3235 253 191 132 127 4 71 MVLESILRR Her2/neu.889.V2 216 273 207 153 22 5 72 MVLESILRK Her2/neu.889.V2K9 61 16 — 2636 381 3 73 LVSEFSRMAR Her2/neu.972 1528 182 49 126 36 4 74 LVSEFSRMAK Her2/neu.972.K10 250 71 2250 5273 62 3 75 ASPLDSTFYR Her2/neu.997 — 90 150 2071 154 3 ¹A positive response is defined as 10% specific lysis above background in the ⁵¹ Cr release assay or twice background and at least 50 pg IFNγ/well above background in at least one of 48 wells tested. ²The tumor target cell lines used are SW480 (A3−, CEA+) and SW403 (A3+, CEA+). Only tumor target data from wells positive for recognition of peptide-pulsed target cells is shown. ³— indicates binding affinity = 10,000 nM.

TABLE 16c Mage2/3-derived A3 supertype crossbinders SEQ Number of pulsed Tumor² ID HLA B9inding (IC₅₀ nM) A3 alleles EHM target target NO Sequence Source A*0301 A*1101 A*3101 A*3301 A*6801 crossbound cell line cell line 76 SSFSTTINY MAGE2.69 1158 107 2727 1526 727 1 77 SSFSTTINK MAGE2.69.K9 69 3.0 2195 —³ 26 3 78 SVFSTIINK MAGE2.69.V2R9 20 8.2 3333 9667 5.7 3 79 SVFSTTINR MAGE2.69.V2R9 58 6.3 62 88 6.7 5 80 FSTINYILWR MAGE2.71 1000 353 257 3919 163 3 81 STINYTLWK MAGE2.72.K10 126 9.2 — — 258 3 82 TTINYTLWR MAGE2.73 204 11 237 171 17 5 7/7 2/5 83 TVINYTLWR MAGE2.73.V2 262 77 720 433 15 4 84 TVINYTLWK MAGE2.73.V2K9 306 97 9000 — 62 3 85 LVHFLLLK MAGE2/3.116 379 40 — — 400 3 86 LVHFLLLKY MAGE2/3.116 297 500 — 8788 8000 2 87 LVHFLLLKK MAGE2/3.116.K9 21 4.3 — — 381 3 0/1 0/1 88 LVHFLLLKLR MAGE2/3.116.R9 440 375 237 94 27 5 89 SMLEVFEGR MAGE2.226 5500 273 37 9.0 1818 3 90 SMLEVFEGK MAGE2.226.K9 116 3.8 120 387 2581 4 91 SVFAHPRK MAGE2.237 78 74 1385 — 182 3 92 ALIEISYVK MAGE2.277 136 32 900 — 286 3 93 AVIETSYVK MAGE2.277.V2 393 63 — — 31 3 94 AVIETSYVR MAGE2.277.V2R9 — 171 129 1160 15 3 95 ISYPPLHER MAGE2.299 324 214 23 36 81 5 96 IVYPPLHER MAGE2.299.V2 117 375 95 32 14 5 97 IVYPPLHEK MAGE2.299.V2K9 42 103 857 2990 42 3 98 YFFPVIFSK MAGE3.138 5000 462 316 207 571 3 99 YVFPVIFSK MAGE3.138.V2 24 3.0 2769 784 1.7 3 100 YVFPVIFSR MAGE3.138.V2R9 36 2.6 6.0 13 0.50 5 101 LLGDNQIMPK MAGE3.189 500 375 — — 372 3 102 SVLEVFEGR MAGE3.226 — 43 106 44 93 4 103 SVLEVFEGK MAGE3.226.K9 83 6.7 129 460 186 5 SEQ ID Immunogenicity Published NO Sequence Peptide Wildtype Tumor Peptide Tumor 76 SSFSTTINY 77 SSFSTTINK 78 SVFSTIINK 79 SVFSTTINR 80 FSTINYILWR 3/3 0/2 + + 81 STINYTLWK 82 TTINYTLWR 83 TVINYTLWR 84 TVINYTLWK 85 LVHFLLLK 86 LVHFLLLKY + nt 87 LVHFLLLKK 0/1 0/1 0/1 88 LVHFLLLKLR 89 SMLEVFEGR 90 SMLEVFEGK 91 SVFAHPRK 92 ALIEISYVK 93 AVIETSYVK 94 AVIETSYVR 95 ISYPPLHER 96 IVYPPLHER 97 IVYPPLHEK 98 YFFPVIFSK 99 YVFPVIFSK 100 YVFPVIFSR 101 LLGDNQIMPK + nt 102 SVLEVFEGR 103 SVLEVFEGK ¹A positive response is defined as 10% specific lysis above bacground in the ⁵¹Cr release assay or twice background and at least 50 pg IFNg/well above background in at least one of 48 wells tested. ²The tumor target cell lines used are 938mel (A3−, Mage2+, Mage3+) and 624mel (A3+, Mage2+, Mage3+). Only tumor target data from wells positive for recognition of peptide-pulsed target cells is shown. ³— indicates binding affinity = 10,000 nM

TABLE 16d p53-derived A3 supertype crossbinders SEQ Tumor² Number of pulsed target ID HLA Binding (IC₅₀ nM) A3 alleles EHM target cell NO Sequence Source A*0301 A*1101 A*3101 A*3301 A*6801 crossbound cell line line 104 KTYQGSYGFK p53.101.K10 22 14 129 —³ 67 4 0/1 0/1 105 KVYQGSYGFR p53.101.V2 38 62 72 — 40 4 106 KVYQGSYGFK p53.101.V2K10 33 9.2 138 — 38 4 107 CTYSPALNK p53.124 24 5.5 1500 518 36 3 0/1 nt 108 BVYSPALNK⁴ p53.124.B1V2 16 13 439 — 500 4 109 BVYSPALNR⁴ p53.124.B1V2R9 25 8.3 33 85 15 5 110 KMPCQLAK p53.132 29 17 353 — 727 3 111 GTRVRAMAIYK p53.154 10 18 16 — 533 3 0/1 nt 112 GVRVRAMAIYK p53.154.V2 58 136 419 — — 3 113 RVRAMAIYK p53.156 7.3 8.2 5.0 4603 2667 3 114 RVRAMAIYR p53.156.R9 41 1667 9.0 138 667 3 115 VVRRCPHHER p53.172 111 3529 ND ND ND 1 116 VVRRHPHHEK⁴ p53.172.B5K10 61 29 196 — 3810 3 1/2 1/2 117 SSCMGGMNR p53.240 550 4.3 ND ND ND 1 118 SSBMGGMNK⁴ p53.240.B3K9 20 5.5 — — 140 3 119 SVBMGGMNR⁴ p53.240.V2B3 162 95 120 853 11 4 120 SVBMGGMNK⁴ p53.240.V2B3K9 13 17 9000 — 30 3 121 SSCMGGMNRR p53.240 — 70 ND ND ND 1 122 SSBMGGMNRK⁴ p53.240.B3K10 262 38 720 — 103 3 123 SVBMGGMNRK⁴ p53.240.V2B3K10 100 75 — — 17 3 124 RVCACPGR p53.273 31 122 206 193 571 4 0/1 nt 125 RVCACPGRDRR p53.273 379 207 346 — 667 3 126 STSRHKKLMFK p53.376 36 46 295 — 533 3 1/2 nt 127 SVSRHKKLMFK p53.375.V2 33 55 295 — 1509 3 128 SVSRHKKLMFR p53.376.V2R11 196 2857 184 1381 500 3 SEQ ID Immunogenicity Published NO Sequence Peptide Wildtype Tumor Peptide Tumor 104 KTYQGSYGFK 2/2 0/1 0/1 105 KVYQGSYGFR 106 KVYQGSYGFK 107 CTYSPALNK 0/1 nt 108 BVYSPALNK⁴ 109 BVYSPALNR⁴ 110 KMPCQLAK 111 GTRVRAMAIYK 0/1 nt 112 GVRVRAMAIYK 113 RVRAMAIYK 114 RVRAMAIYR 115 VVRRCPHHER 116 VVRRHPHHEK⁴ 2/3 1/2 1/2 + + 117 SSCMGGMNR 118 SSBMGGMNK⁴ 119 SVBMGGMNR⁴ 120 SVBMGGMNK⁴ 121 SSCMGGMNRR 0/1 nt nt 122 SSBMGGMNRK⁴ 123 SVBMGGMNRK⁴ 124 RVCACPGR 0/1 nt nt 125 RVCACPGRDRR 126 STSRHKKLMFK 1/2 nt 127 SVSRHKKLMFK 128 SVSRHKKLMFR ¹A positive response is defined as 10% specific lysis above bacground in the ⁵¹Cr release assay or twice background and at least 50 pg IFNg/well above background in at least one of 48 wells tested. ²The tumor target cell lines used are SW403 (A3+, p53−) and SW403/p53 transfectant (A3+, p53+). Only tumor target data from wells positive for recognition of peptide-pulsed target cells is shown. ³— indicates binding affinity = 10,000 nM. ⁴B = α-amino butyric acid. α-amino butryic acid may be replaced with cysteine.

TABLE 17a CEA-derived B7 supertype crossbinders SEQ Number of ID B*0702 B*3501 B*5101 B*5301 B*5401 B7 alleles Immunogenicity NO Sequence Source nM nM nM nM nM crossbound Peptide Wildtype Tumor 129 IPWQRLLLTA CEA.13 125 — 2115 2657 3.2 2 130 IPWQRLLLTI CEA.13I10 39 — 19 291 270 4 0/1 131 LPQHLFGYSW CEA.58 — 9000 — 22 4348 1 132 LPQHLFGYSI CEA.58I10 212 — 262 930 172 3 133 YPNASLLI CEA.102 196 514 8.1 40 137 4 — indicates binding affinity = 10,000 nM.

TABLE 17b Her2/neu-derived B7 supertype crossbinders SEQ Number of ID B*0702 B*3501 B*5101 B*5301 B*5401 B7 alleles Immunogenicity NO Sequence Source nM nM nM nM nM crossbound Peptide Wildtype Tumor 134 KPYDGIPA Her2/neu921 367 — — 490 29 3 135 KPYDGIPI Her2/neu.921I8 115 — 74 51.67 303 3 136 LPQPPICTI Her2/neu.941 196 — 4.2 28 19 4 137 PPSPREGPLPA Her2/neu.1149 12 — — — 83 2 138 PPSPREGPLPI Her2/neu.1149.I11 22 — 423 85 — 3 1/2 0/2 139 SPAFDNLYI Her2/neu.1214 290 — 229 155 — 3 — indicates binding affinity = 10,000 nM.

TABLE 17c Mage2/3-derived B7 supertype crossbinders SEQ Number of ID B*0702 B*3501 B*5101 B*5301 B*5401 B7 alleles NO Sequence Source nM nM nM nM nM crossbound 140 VPISHLYIL MAGE2.170 22 171 96 239 3125 4 141 VPISHLYAL MAGE2.170.A8 23 195 135 6643 8333 3 142 VPISMLYIL MAGE2.170.M5 164 274 70 1069 1493 3 143 VPISHLYILV MAGE2.170 2037 — 42 5471 100 2 144 VPISHLYILI MAGE2.170.I10 367 2667 50 169 2222 3 145 LPTTMNYPL MAGE3.71 68 23 1964 266 2564 3 146 LPTTMNYPI MAGE3.7LI9 100 343 31 182 4.2 5 147 YPLWSQSY MAGE3.77 — 450 >9167 — 7692 1 148 YPLWSQSI MAGE3.77.I8 60 3790 5.8 258 238 4 149 MPKAGLLI MAGE3.196 42 — 12 358 313 4 150 MPKAGLLII MAGE3.196 932 5143 393 90 248 3 151 MPVAGLLII MAGE3.196.V3 86 66 1.2 2.3 112 5 152 MPKAGLLIIV MAGE3.196 1774 — 393 — 12 2 153 MPKAGLLIII* MAGE3.196.I10 324 2400 62 176 102 4 SEQ ID Immunogenicity Published NO Sequence Peptide Wildtype Tumor Peptide Tumor 140 VPISHLYIL 6/6 0/6 141 VPISHLYAL 142 VPISMLYIL 143 VPISHLYILV 144 VPISHLYILI 145 LPTTMNYPL 146 LPTTMNYPI 147 YPLWSQSY 148 YPLWSQSI 149 MPKAGLLI 150 MPKAGLLII + nt 151 MPVAGLLII 152 MPKAGLLIIV 153 MPKAGLLIII* *This 10mer may also be used as a 9mer (MPKAGLLII) (SEQ ID NO:362) — indicates binding affinity = 10,000 nM.

TABLE 17d p53-derived B7 supertype crossbinders SEQ Number of ID B*0702 B*3501 B*5101 B*5301 B*5401 B7 alleles Immunogenicity NO Sequence Source nM nM nM nM nM crossbound Peptide Wildtype Tumor 154 APAAPTPAA p53.76 18 360 — — 1.9 3 155 APAAPTPAAPA p53.76 14 32.73 — 93 53 3 0/1 nt 156 APAPAPSW p53.84 500 32.73 — 135 91 3 157 APAPAPSI p53.84.18 275 — 500 291 — 3 — indicates binding affinity = 10,000 nM.

TABLE 18a CEA-derived A1 binders SEQ ID A*0101 NO AA Sequence Source nM 158 11 RVDGNRQIIGY CEA.72 294 159 9 QQATPGPAY CEA.87 — 160 9 QQDTPGPAY CEA.87.D3 57 161 11 RSDSVILNVLY CEA.225 47 162 10 PTISPLNTSY CEA.240 1000 163 10 PTDSPLNTSY CEA.240.D3 266 164 9 AASNPPAQY CEA.261 — 165 9 AADNPPAQY CEA.261.D3 46 166 9 ITVNNSGSY CEA.289 2500 167 9 LTDNNSGSY CEA.289.D3 96 168 9 VTRNDVGPY CEA.383 — 169 9 VTDNDVGPY CEA.383.D3 4.1 170 11 HSDPVILNVLY CEA.403 26 171 10 PTISPSYTYY CEA.418 325 172 10 PTDSPSYTYY CEA.418.D3 1.1 173 9 PTISPSYTY CEA.418 7143 174 9 PTDSPSYTY CEA.418.D3 38 175 9 TISPSYTYY CEA.419 1042 176 9 TIDPSYTYY CEA.419.D3 3.1 177 10 HAASNPPAQY CEA.438 2688 178 9 AADNPPAQY CEA.439.D3 45 179 9 ITEKNSCLY CEA.467 641 180 9 ITDKNSGLY CEA.467.D3 12 181 11 RSDPVTLDVLY CEA.581 7.8 182 10 HSASNPSPQY CEA.616 74 183 10 HTASNFSPQY CEA.616.T2 132 184 10 HSDSNFSPQY CEA.616.D3 45 — indicates binding affinity = 10,000 nM.

TABLE 18b Her2/neu-derived A1 binders SEQ A*0101 ID NO AA Sequence Source nM 185 9 VMAGVGSPY Her2/neu.773 625 186 9 VMDGVGSPY Her2/neu.773.D3 40 187 10 CMQIAKGMSY Her2/neu.826 83 188 10 CTQIAKGMSY Her2/neu.826.T2 19 189 9 LLDIDETEY Her2/neu.869 3.3 190 9 LTDIDETEY Her2/neu.869.T2 5.7 191 10 FTHQSDVWSY Her2/neu.899 9.3 192 10 FTDQSDVWSY Her2/neu.899.D3 0.60 193 10 PASPLDSTFY Her2/neu.996 1667 194 10 PADPLDSTFY Her2/neu.996.D3 19 195 9 ASPLDSTFY Her2/neu.997 862 196 9 ATPLDSTFY Her2/neu.997.T2 36 197 10 MGDLVDAEEY Her2/neu.1014 2083 198 10 MTDLVDAEEY Her2/neu.1014.T2 2.3 199 9 LTCSPQPEY Her2/neu.1131 192 200 9 LTDSPQPEY Her2/neu.1131.D3 32 201 10 FSPAFDNLYY Her2/neu.1213 4.5 212 10 FTPAFDNLYY Her2/neu.1213.T2 0.80 203 9 FSPAFDNLY Her2/neu.1213 581 204 9 FTPAFDNLY Her2/neu.1213.V2 7.8 205 9 SPAFDNLYY Her2/neu.1214 NT 206 9 SPDFDNLYY Her2/neu.1214.D3 74 207 10 GTPTAENPEY Her2/neu.1239 397 208 10 GTDTAENPEY Her2/neu.1239.D3 26 — indicates binding affinity = 10,000 nM.

TABLE 18c Mage2/3-derived A1 binders SEQ Published ID A*0101 Immunogenicity NO AA Sequence Source nM Peptide Tumor 209 10 ASSFSTTINY MAGE2.68 1563 210 10 ATSFSTTINY MAGE2.68.T2 455 211 10 ASDFSTTINY MAGE2.68.D3 25 212 9 SSFSTTINY MAGE2.69 1563 213 9 STFSTTINY MAGE2.69.T2 490 214 11 VVEVVPISHLY MAGE2.166 125 215 8 VTCLGLSY MAGE2.179 1136 216 8 VTDLGLSY MAGE2.179.D3 2.7 217 10 LMQDLVQENY MAGE2.246 556 218 10 LTQDLVQENY MAGE2.246.T2 58 219 9 MQDLVQENY MAGE2.247 17 220 9 MTDLVQENY MAGE2.247.T2 0.80 221 10 ASSLPTTMNY MAGE3.68 11 222 10 ATSLPTTMNY MAGE3.68.T2 208 223 10 ASDLPTTMNY MAGE3.68.D3 2.6 224 9 SSLPTTMNY MAGE3.69 676 225 9 STLPTTMNY MAGE3.69.T2 58 226 11 TMNYPLWSQSY MAGE3.74 301 227 9 GSVVGNWQY MAGE3.137 4237 228 9 GTVVGNWQY MAGE3.137.T2 36 229 11 LMEVDPIGHLY MAGE3.166 3.3 230 9 EVDPIGHLY MAGE3.168 6.8 + + 231 9 ETDPIGHLY MAGE3.168.T2 0.70 232 8 ATCLGLSY MAGE3.179 227 233 10 LTQEFVQENY MAGE3.246 96 234 10 LTDHFVQENY MAGE3.246.D3 2.3 235 9 ISGGPHISY MAGE3.293 676 236 9 ITGGPHISY MAGE3.293.T2 36

TABLE 18d p53-derived A1 binders SEQ ID A*0101 NO AA Sequence Source nM 237 10 PSQKTYQGSY p53.98 1786 238 10 PTQKTYQGSY p53.98.T2 36 239 10 GTAKSVTCTY p53.117 76 240 10 GTDKSVTCTY p53.117.D3 42 241 10 RVEGNLRVEY p53.196 1136 242 10 RVDGNLRVEY p53.196.D3 46 243 10 VGSDCTTIHY p53.225 96 244 9 GSDCTTIHY p53.226 0.80 245 9 GTDCTTIHY p53.226.T2 0.90 246 11 GSDCTTIHYNY p53.226 68

TABLE 19a CEA-derived A24 binders SEQ Published ID A*0101 Immunogenicity NO AA Sequence Source nM Peptide Tumor 247 9 RWCIPWQRL CEA10 923 248 9 RYCIPWQRF CEA.10.Y2F9 191 249 10 RWCIPWQRLL CEA.10 308 250 10 RYCIPWQRLF CEA.10.Y2F10 26 251 11 RWCIPWQRLLL CEA10 152 252 11 PWQRLLLTASL CEA.14 324 253 10 FWNPPTTAKL CEA.27 400 254 10 FYNPPTTAKF CEA.27.Y2F10 182 255 8 IYPNASLL CEA.101 177 256 9 IYPNASLLI CEA.101 1.7 257 9 IYPNASLLF CEA.101.F9 2.2 258 11 FYTLHVIKSDL CEA.119 480 259 10 VYPELPKPSI CEA.140 1519 260 10 VYPELPKPSF CEA.140.F10 106 261 9 LWWVNNQSL CEA.177 546 262 9 LYWVNNQSF CEA.177.Y2F9 63 263 9 LYGPDAPTI CEA.234 57 264 9 LYGPDAPTF CEA.234.F9 63 265 10 QYSWFVNGTF CEA.268 3.5 266 8 SWFVNGTF CEA.270 480 267 10 TFQQSTQELF CEA.276 750 268 10 TYQQSTQELF CEA.276.Y2 308 269 9 VYAEPPKPF CEA.318 41 270 10 VYAEPPKPFI CEA.318 667 271 10 VYAEPPKPFF CEA.318.F10 27 272 11 TYLWWVNNQSL CEA.353 46 273 9 LYGPDDPTI CEA.412 353 274 11 SYTYYRPGVNL CEA.423 218 275 9 TYYRPGVNL CEA.425 185 276 9 TYYRPGVNF CEA.425.F9 52 277 11 TYYRPGVNLSL CEA.425 132 278 10 YYRPGVNLSL CEA.426 86 279 10 YYRPGVNLSF CEA.426.F10 10 280 10 QYSWLIDGNI CEA.446 800 281 10 QYSWLIDGNF CEA.446.F10 60 282 11 TYLWWVNGQSL CEA.531 92 283 9 LWWYNGQSL CEA.533 1463 284 9 LYWVNGQSF CEA.533.Y2F9 16 285 9 LYGPDTPII CEA.590 46 286 10 SYLSGANLNL CEA.604 207 287 10 SYLSGANLNF CEA.604.F10 10 288 9 QYSWRINGI CEA.624 444 289 9 QYSWRINGF CEA.624.F9 109 290 9 TYACFVSNL CEA.652 10 + + 291 9 TYACFVSNF CEA.652.F9 8.6

TABLE 19b Her2/neu-derived A24 binders SEQ ID A*2401 NO AA Sequence Source nM 292 9 PYVSRLLGI Her2/neu.780 71 293 9 PYVSRLLGF Her2/neu.780.F9 9.2 294 11 PYVSRLLGICL Her2/neu.780 375 295 10 GMSYLEDVRL Her2/neu.832 NT 296 10 GYSYLEDVRF Her2/neu.832.Y2F10 235 297 9 KWMALESIL Her2/neu.887 800 298 9 KYMALESIF Her2/neu.887.Y2F9 19 299 9 RFTHQSDVW Her2/neu.898 1091 300 9 RYTHQSDVF Her2/neu.898.Y2F9 60 301 9 VWSYGVTVW Her2/neu.905 150 302 9 VYSYGVTVF Her2/neu.905.Y2F9 16 303 11 VWSYGVTVWEL Her2/neu.905 130 304 9 SYGVTVWEL Her2/neu.907 100 305 9 SYGVTVWEF Her2/neu.907.F9 26 306 9 VYMIMVKCW Her2/neu.951 75 307 9 VYMTMVKCF Her2/neu.951.F9 19 308 11 VYMIMVKCWMI Her2/neu.951 6.7 309 9 RFRELVSEF Her2/neu.968 667 310 9 RYRELVSEF Her2/neu.968.Y2 36 311 9 RMARDPQRF Her2/neu.978 3750 312 9 RYARDPQRF Her2/neu.978.Y2 120

TABLE 19c Mage2/3-derived A24 binders SEQ Published ID A*2401 Immunogenicity NO AA Sequence Source nM Peptide Tumor 313 11 SFSTTINYTLW MAGE2.70 429 314 10 SYSTTINYTF MAGE2.70.Y2F10 15 315 9 MFPDLESEF MAGE2.97 857 316 9 MYPDLESEF MAGE2.97.Y2 52 317 9 KMVELVHFL MAGE2.112 750 318 9 KYVELVHFF MAGE2.112.Y2F9 7.1 319 11 IFSKASEYLQL MAGE2.150 126 320 9 IYSKASEYF MAGE2.150.Y2F9 15 321 9 EYLQLVFGI MAGE2.156 3.4 + + 322 9 EYLQLVFGF MAGE2.156.F9 4.0 323 10 LYILVTCLGL MAGE2.175 857 324 10 LYILVTCLGF MAGE2.175.F10 18 325 9 VMPKTGLLI MAGE2.195 52 326 9 VYPKTGLLF MAGE2.195.Y2F9 5.5 327 10 VMPKTGLLII MAGE2.195 207 328 10 VYPKTGLLIF MAGE2.195.Y2F10 2.9 329 10 EFLWGPRALI MAGE2.270 1237 330 10 EYLWGPRALF MAGE2.270.Y2F10 10 331 8 LWGPRALI MAGE2.272 100 332 10 SYVKVLHHTL MAGE2.282 75 333 10 SYVKVLHHTF MAGE2.282.F10 34 334 9 TFPDLESEF MAGE3.97 2449 + + 335 9 TYPDLESEF MAGE3.97.Y2 218 336 9 NWQYFFPVI MAGE3.142 23 337 9 NYQYFFPVF MAGE3.142.Y2F9 3.4 338 10 NYQYFFPVIF MAGE3.142.Y2 23 339 8 QYFFPVIF MAGE3.144 100 340 9 IFSKASSSL MAGE3.150 750 341 9 IYSKASSSF MAGE3.150.Y2F9 375 342 11 IFSKASSSLQL MAGE3.150 132 343 10 LYIFATCLGL MAGE3.175 3429 344 10 LYIFATCLGF MAGE3.175.F10 10 345 9 IMPKAGLLI MAGE3.195 29 + + 346 9 IYPKAGLLF MAGE3.195.Y2F9 9.2 347 10 IMPKAGLLII MAGE3.195 240 348 10 IYPKAGLLIF MAGE3.195.Y2F10 1.2 349 11 IWEELSVLEVF MAGE3.221 462 350 8 SYPPLHEW MAGE3.300 286 351 10 SYPPLHEWVL MAGE3.300 20 352 10 SYPPLHEWVF MAGE3.300.F10 5.5

TABLE 19d p53-derived A24 binders SEQ ID A*2401 NO AA Sequence Source nM 353 9 TFSDLWKLL p53.18 — 354 9 TYSDLWKLF p53.18.Y2F9 5.5 355 8 TYQGSYGF p53.102 109 356 10 TYQGSYGFRL p53.102 100 357 10 TYQGSYGFRF p53.102.F10 30 358 8 SYGFRLGF p53.106 429 359 9 SYGFRLGFL p53.106 600 360 9 SYGFRLGFF p53.106.F9 121 361 10 TYSPALNKMF p53.125 2.4 — indicates binding affinity = 10,000 nM

TABLE 20 TAA A3 supertype candidates SEQ No. A3 supertype ID A*0301 A*1101 A*3101 A*3301 A*6801 alleles crossbound NO Sequence Source nM nM nM nM nM (200) 42 HLFGYSWYK CEA.61 2.2 2.4 20 18 3.5 5 44 TISPLNTSYK CEA.241.K10 61 182 — — 116 3 46 RTLTLLSVTR CEA.376 524 55 6.0 1036 160 3 51 IVPSYTYYR CEA.420.V2 92 13 26 58 2.6 5 52 RTLTLFNVTR CEA.554 111 13 5.0 1611 99 4 54 HTQVLFIAK CEA.636 1183 35 106 132 160 4 55 FVSNTLATGR CEA.656 5790 122 333 104 8.2 3 57 VVFGILIKR Her2/neu.669 100 8.3 13 78 4.0 5 60 KIRKYTMRR Her2/neu.681 15 3333 16 4028 — 2 62 VLRENTSPK Her2/neu.754 28 462 129 290 — 2 67 LVKSPNHVK Her2/neu.852 23 86 182 784 73 4 68 KITDFGLAR Her2/neu.860 65 25 100 — 1633 3 69 KVTDFGLAR Her2/neu.860.V2 201 76 106 — 133 3 70 MALESILRR Her2/neu.889 3235 253 191 132 127 3 73 LVSEFSRMAR Her2/neu.972 1528 182 49 126 36 4 75 ASPLDSTFYR Her2/neu.997 — 90 150 2071 154 3 77 SSFSTTINK MAGE2.69.K9 69 3 2195 — 26 3 82 TTINYTLWR MAGE2.73 204 11 237 171 17 3 90 SMLEVFEGK MAGE2.226.K9 116 3.8 120 387 2581 3 91 SVFAHPRK MAGE2.237 78 74 1385 — 182 3 96 IVYPPLHER MAGE2.299.V2 117 375 95 32 14 4 99 YVFPVIFSK MAGE3.138.V2 24 3.0 2769 784 1.7 3 103 SVLEVFEGK MAGE3.226.K9 83 6.7 129 460 186 4 104 KTYQGSYGFK p53.101.K10 22 14 129 — 67 4 107 CTYSPALNK p53.124 24 5.5 1500 518 36 3 111 GTRVRAMAIYK p53.154 10 18 16 — 533 3 116 VVRRBPHHEK* p53.172.B5K10 61 29 196 — 3810 3 119 SVBMGGMNR* p53.240.V2B3 162 95 120 853 11 4 124 RVCACPGR p53.273 31 122 106 193 571 4 SEQ ID Immunogenicity Published NO Sequence Peptide Wildtype Tumor Peptide Tumor 42 HLFGYSWYK 3/4 2/4 + + 44 TISPLNTSYK 3/3 2/3 0/1 46 RTLTLLSVTR 51 IVPSYTYYR 52 RTLTLFNVTR 1/1 nt 54 HTQVLFIAK 55 FVSNTLATGR 57 VVFGILIKR 1/3 0/1 60 KIRKYTMRR 3/3 2/3 + + 62 VLRENTSPK 4/7 3/6 + + 67 LVKSPNHVK 0/3 0/1 + − 68 KITDFGLAR 0/1 nt 69 KVTDFGLAR 70 MALESILRR 73 LVSEFSRMAR 75 ASPLDSTFYR 77 SSFSTTINK 82 TTINYTLWR 3/3 0/2 + + 90 SMLEVFEGK 91 SVFAHPRK 96 IVYPPLHER 99 YVFPVIFSK 103 SVLEVFEGK 104 KTYQGSYGFK 2/2 0/1 0/1 107 CTYSPALNK 0/1 nt 111 GTRVRAMAIYK 0/1 nt 116 VVRRBPHHEK* 2/3 1/2 1/2 + + 119 SVBMGGMNR* 124 RVCACPGR 0/1 nt nt — indicates binding affinity ≧10,000 nM. *B = α-amino butyric acid. In some embodiments, α-amino butyric acid may be replaced with cysteine

TABLE 21 TAA-derived B7 supertype candidates No. B7 supertype SEQ alleles ID B*0702 B*3501 B*5101 B*5301 B*5401 crossbound NO Sequence Source nM nM nM nM nM (200) 133 YPNASLLI CEA.102 196 514 8.1 40 137 4 136 LPQPPICTI Her2/neu.941¹ 196 —² 4.2 28 19 4 140 VPISHLYIL MAGE2.170 22 171 96 239 3125 3 146 LPITMNYPI MAGE3.71.I9 100 343 31 182 4.2 4 153 MPKAGLLIII¹ MAGE3.196.I10 324 2400 62 176 102 3 155 APAAPTPAAPA p53.76 14 3273 — 93 53 3 SEQ ID Immunogenicity Published NO Sequence Peptide Wildtype Tumor Peptide Tumor 133 YPNASLLI 136 LPQPPICTI 140 VPISHLYIL 6/6 0/6 146 LPITMNYPI 153 MPKAGLLIII¹ + 155 APAAPTPAAPA 0/1 nt ¹This 10mer may also be used as a 9mer (MPKAGLLII) (SEQ ID NO:362) ²— indicates binding affinity = 10,000 nM.

TABLE 22 TAA-derived A1 candidates SEQ Published ID A*0101 Immunogenicity NO AA Sequence Source nM Peptide Tumor 161 11 RSDSVILNVLY CEA.225 47 167 9 ITDNNSGSY CEA.289.D3 96 170 11 HSDPVILNVLY CEA.403 26 172 10 PTDSPSYTYY CEA.418.D3 1.1 178 9 AADNPPAQY CEA.439.D3 45 180 9 ITDKNSGLY CEA.467.D3 12 181 11 RSDPVTLDVLY CEA.581 7.8 182 10 HSASNPSPQY CEA.616 74 186 9 VMDGVGSPY Her2/neu.773.D3 40 188 10 CTQIAKGMSY Her2/neu.826.T2 19 189 9 LLDIDETEY Her2/neu.869 3.3 191 10 FTHQSDVWSY Her2/neu.899 9.3 194 10 PADPLDSTFY Her2/neu.996.D3 19 198 10 MTDLVDAEEY Her2/neu.1014.T2 2.3 200 9 LTDSPQPEY Her2/neu.1131.D3 32 201 10 FSPAFDNLYY Her2/neu.1213 4.5 208 10 GTDTAENPEY Her2/neu.1239.D3 26 211 10 ASDFSTTINY MAGE2.68.D3 25 216 8 VTDLGLSY MAGE2.179.D3 2.7 219 9 MQDLVQENY MAGE2.247 17 221 10 ASSLPTTMNY MAGE3.68 11 228 9 GTVVGNWQY MAGE3.137.T2 36 230 9 EVDPIGHLY MAGE3.168 6.8 + + 234 10 LTDHFVQENY MAGE3.246.D3 2.3 236 9 ITGGPHISY MAGE3.293.T2 36 238 10 PTQKTYQGSY p53.98.T2 36 239 10 GTAKSVTCTY p53.117 76 240 10 GTDKSVTCTY p53.117.D3 42 242 10 RVDGNLRVEY p53.196.D3 46 246 11 GSDCTTIHYNY p53.226 68

TABLE 23 TAA-derived A24 candidates SEQ Published ID A*0101 Immunogenicity NO AA Sequence Source nM Peptide Tumor 256 9 IYPNASLLI CEA.101 1.7 263 9 LYGPDAPTI CEA.234 57 265 10 QYSWFVNGTF CEA.268 3.5 269 9 VYAEPPKPF CEA.318 41 272 11 TYLWWVNNQSL CEA.353 46 278 10 YYRPGVNLSL CEA.426 86 279 10 YYRPGVNLSF CEA.426.F10 10 281 10 QYSWLIDGNF CEA.446.F10 60 282 11 TYLWWYNGQSL CEA.531 92 285 9 LYGPDTPII CEA.590 46 287 10 SYLSGANLNF CEA.604.F10 10 290 9 TYACFVSNL CEA.652 10 292 9 PYVSRLLGI Her2/neu.780 71 293 9 PYVSRLLGF Her2/neu.780.F9 9.2 304 9 SYGVTVWEL Her2/neu.907 100 305 9 SYGVTVWEF Her2/neu.907.F9 26 308 11 VYMIMVKCWMI Her2/neu.951 6.7 310 9 RYRELYSEF Her2/neu.968.Y2 36 316 9 MYPDLESEF MAGE2.97.Y2 52 321 9 EYLQLVFGI MAGE2.156 3.4 324 10 LYILVTCLGF MAGE2.175.F10 18 325 9 VMPKTGLLI MAGE2.195 52 331 8 LWGPRALI MAGE2.272 100 332 10 SYVKVLHHTL MAGE2.282 75 333 10 SYVKVLHHTF MAGE2.282.F10 34 334 9 TFPDLESEF MAGE3.97 2449 335 9 TYPDLESEF MAGE3.97.Y2 218 336 9 NWQYFFPVI MAGE3.142 23 344 10 LYIFATCLGF MAGE3.175.F10 10 345 9 IMPKAGLLI MAGE3.195 29 351 10 SYPPLHEWVL MAGE3.300 20 356 10 TYQGSYGFRL p53.102 100 361 10 TYSPALNKMF p53.125 2.4

TABLE 24 TAA Candidate Summary A3 supertype B7 supertype A1 A24 Antigen Wildtype Analog Wildtype Analog Wildtype Analog Wildtype Analog Total CEA 5 2 1 0 4 3 9 2 26 Her2/neu 7 1 1 0 3 6 3 1 22 Mage2/3 3 3 1 2 4 4 7 3 27 p53 2 4 1 0 2 2 2 0 13 Total 17 10 4 2 13 15 21 6 88

TABLE 25 Tumor-associated antigen (TAA) sequences CEA SEQ ID NO:363      MESPSAPPHRWCIPWQRLLLTASLLTFWNPPTTAKLTIESTPFNVAEGK EVLLLVHNLPQHLFGYSWYKGERVDGNRQIIGYVIGTQQATPGPAYSGREIIY PNASLLIQNIIQNDTGFYTLHVIKSDLVNEEATGQFRVYPELPKPSISSNNSKPV EDKDAVAFTCEPETQDATYLWWVNNQSLPVSPRLQLSNGNRTLTLFNVTRN DTASYKCETQNPVSARRSDSVILNVLYGPDAPTISPLNTSYRSGENLNLSCHA ASNPPAQYSWFVNGTFQQSTQELFIPNITVNNSGSYTCQAHNSDTGLNRTTVT TITVYAEPPKPFITSNNSNPVEDEDAVALTCEPEIQNTTYLWWVNNQSLPVSPR LQLSNDNRTLTLLSVTRNDVGPYECGIQNELSVDHSDPVILNVLYGPDDPTISP SYTYYRPGVNLSLSCHAASNPPAQYSWLIDGNIQQHTQELFISNITEKNSGLYT CQANNASASGHSRTTVKTITVSAELPKPSISSNNSKPVEDKDAVAFTCEPEAQN TTYLWWVNGQSLPVSPRLQLSNGNRTLTLFNVTRNDARAYVCGIQNSVSAN RSDPVTLDVLYGPDTPIISPPDSSYLSGANLNLSCHSASNPSPQYSWRINGIPQQ HTQVLFIAKITPNNNGTYACFVSNLATGRNNSIVKSITVSASGTSPGLSAGATV GIMIGVLVGVALI Her2/neu SEQ ID NO:364      MELAALCRWGLLLALLPPGAASTQVCTGTDMKLRLPASPETHLDMLR HLYQGCQVVQGNLELTYLPTNASLSFLQDIQEVQGYVLIAHNQVRQVPLQRL RIVRGTQLFEDNYALAVLDNGDPLNNTTPVTGASPGGLRELQLRSLTEILKGG VLIQRNPQLCYQDTILWKDIFHKNNQLALTLIDTNRSRACHPCSPMCKGSRC WGESSEDCQSLTRTVCAGGCARCKGPLPTDCCHEQCAAGCTGPKHSDCLAC LHFNHSGICELHCPALVTYNTDTFESMPNPEGRYTFGASCVTACTYNYLSTDV GSCTLVCPHNQEVTAEDGTQRCEKCSKPCARVCYGLGMEHLREVRAVTSA NIQEFAGCKKIFGSLAFLPESFDGDPASNTAPLQPEQLQVFETLEEITGYLYISA WPDSLPDLSVFQNLQVIRGRILHNGAYSLTLQGLGISWLGLRSLRELGSGLALI HHNTHLCFVHTVPWDQLFRNPHQALLHTANRPEDECVGEGLACHQLCARGH CWGPGPTQCVNCSQFLRGQEVEECRVLQGLPREYVNARHCLPCHPECQPQ NGSVTCFGPEADQCVACAHYKDPPFCVARCPSGVKPDLSYMPIWKFPDEEGA CQPCPINCTHSCVDLDDKGCPAEQRASPLTSIISAVVGILLVVVLGVVFGILIKR RQQKIRKYTMRRLLQETELVEPLTPSGAMPNQAQMRILKETELRKVKVLGSG AFGTVYKGIWIPDGENVKIPVAIKVLRENTSPKANKEILDEAYVMAGVGSPYV SRLLGICLTSTVQLVTQLMPYGCLLDHVRENRGRLGSQDLLNWCMQIAKGM SYLEDVRLVHRDLAARNVLKSPNHVKITDFGLARLLDIDETEYHADGGKVP IKWMALESILRRRFTHQSDVWSYGVTVWELMTFGAKPYDGIPAREIPDLLEK GERLPQPPICTIDVYMIMVKCWMIDSECRPRFRELVSEFSRMARDPQRFVVIQ NEDLGPASPLDSTFYRSLLEDDDMGDLVDAEEYLVPQQGFFCPDPAPGAGGM VHHRHRSSSTRSGGGDLTLGLEPSEEEAPRSPLAPSEGAGSDVFDGDLGMGA AKGLQSLPTHDPSPLQRYSEDPTVPLPSETDGYVAPLTCSPQPEYVNPQDVRP QPPSPREGPLPAARPAGATLERPKTLSPGKNGVVKDVFAFGGAVENPEYLTPQ GGAAPQPHPPPAFSPAFDNLYYWDQDPPERGAPPSTFKGTPTAENPEYLGLDV PV MAGE2 SEQ ID NO:365      MPLEQRSQHCKPEEGLEARGEALGLVGAQAPATEEQQTASSSSTLVEV TLGEVPAADSPSPPHSPQGASSFSTTINYTLWRQSDEGSSNQEEEGPRMFPDLE SEFQAAISRKMVELVHFLLLKYRAREPVTKAEMLESVLRNCQDFFPVIFSKAS EYLQLVFGIEVVEVVPISHLYILVTCLGLSYDGLLGDNQVMPKTGLLIIVLAIIA IEGDCAPEEKIWEELSMLEVFEGREDSVFAHPRKLLMQDLVQENYLEYRQVP GSDPACYEFLWGPRALIETSYVKVLHHTLKIGGEPHISYPPLHERALREGEE MAGE3 SEQ ID NO:366      MPLEQRSQHCKPEEGLEARGEALGLVGAQAPATEEQEAASSSSTLVEV TLGEVPAAESPDPPQSPQGASSLPTTMNYPLWSQSYEDSSNQEEEGPSTFPDLE SEFQAALSRKVAELVHFLLLKYRAREPVTKAEMLGSVVGNWQYFFPVIFSKA SSSLQLVFGIELMEVDPIGHLYIFATCLGLSYDGLLGDNQIMPKAGLLIIVLAII AREGDCAPEEKIWEELSVLEVFEGREDSILGDPKKLLTQHFVQENYLEYRQVP GSDPACYEFLWGPRALVETSYVKVLHHMVKISGGPHISYPPLHEWVLREGEE p53 SEQ ID NO:367      MEEPQSDPSVEPPLSQETFSDLWKLLPENNVLSPLPSQAMDDLMLSPD DIEQWFTEDPGPDHEAPRMPEAAPPVAPAPAPAAPTPAAPATAPSWPLSSSVPSQK DIEQWFTEDPGPDEAPRMPEAAPPVAPAPAAPTPAAPAPAPSWPLSSSVPSQK TYQGSYGFRLGFLHSGTAKSVTCTYSPALNKMFCQLAKTCPVQLWVDSTPPP GTRVRAMAIYKQSQHMTEVVRRCPHHERCSDSDGLAPPQHLIRVEGNLRVEY LDDRNTFRHSVVVPYEPPEVGSDCTTIHYNYMCNSSCMGGMNRRPILTIITLE DSSGNLLGRNSFEVRVCACPGRDRRTEEENLRKKGEPHHELPPGSTKRALPN NTSSSPQPKKKPLDEYFTLQIRGRERFEMFRELNEALELKDAQAGKEPGGSR AHSSHLKSKKGQSTSRHKKLMFKTEGPDSD

TAELE 26 CEA-derived B44 peptides Seq ID No. Sequence AA Protein Position B*1801 B*4001 B*4002 B*4402 B*4403 B*4501 Degeneracy 368 AEGKEVLLL 9 CEA 46 359 34 60 179 1.5 161 6 369 QELFIPNITV 10 CEA 282 81 121 27 48 2.6 14 6 370 IESTPFNVAEG 11 CEA 38 87 1074 352 89 8.7 84 5 371 YECGIQNEL 9 CEA 391 82 71 53 452 5.3 855 5 372 YECGIQNELSV 11 CEA 391 9.2 28 26 1714 0.46 155 5 373 MESPSAPPHRW 11 CEA 1 12 943 1915 5.3 41 359 4 374 IESTPFNVA 9 CEA 38 14 2542 36 19,157 1.2 13 4 375 AEGKEVLLLV 10 CEA 46 5135 69 408 223 8.6 994 4 376 KEVLLLVHNL 10 CEA 49 893 1.0 4.4 326 2.3 2512 4 377 REIIYPNASL 10 CEA 98 4340 0.57 7.5 412 1.7 954 4 378 REIIYPNASLL 11 CEA 98 1788 2.4 12 57 0.38 1777 4 379 CETQNPVSA 9 CEA 215 73 7016 261 — 10 15 4 380 QELFIPNIT 9 CEA 282 125 4361 172 1217 3.0 18 4 381 PEAQNTTYLWWV 12 CEA 525 205 3802 1097 414 183 25 4 382 GERVDGNRQII 11 CEA 70 764 278 18 871 1.3 — 3 383 NEEATGQFRVY 11 CEA 131 7.7 3252 999 9.6 69 3986 3 384 CEPETQDAT 9 CEA 167 4009 3646 410 5450 50 97 3 385 GENLNLSCHA 10 CEA 252 14,373 1341 357 8610 5.3 271 3 386 GENLNLSCHAA 11 CEA 252 7838 4557 63 1907 9.0 32 3 387 QELFIPNI 8 CEA 282 127 5815 147 1339 8.5 1319 3 388 CEPEIQNTTYL 11 CEA 345 129 287 1603 1245 60 11,981 3 389 PEIQNTTYLWW 11 CEA 347 172 749 1045 17 227 1365 3 390 PEIQNTTYLWWV 12 CEA 347 517 511 291 167 66 932 3 391 NELSVDHSDPV 11 CEA 397 49 1704 1128 1615 38 78 3 392 QELFISNIT 9 CEA 460 530 6571 58 2334 3.9 80 3 393 PEAQNTTYLWW 11 CEA 525 147 2096 3090 121 79 2005 3 394 GERVDGNRQI 10 CEA 70 9395 1933 49 2544 13 19,464 2 395 NEEATGQFRV 10 CEA 131 998 — — 4536 471 405 2 396 EEATGQFRV 9 CEA 132 611 803 1025 1602 82 13 2 397 EEATGQFRVY 10 CEA 132 64 — 1532 26 1041 1374 2 398 VEDKDAVAF 9 CEA 157 94 121 1583 963 1443 — 2 399 CEPETQDATYL 11 CEA 167 831 311 3388 398 807 — 2 400 VEDEDAVAL 9 CEA 335 840 11 2665 9691 51 — 2 401 CEPEIQNTTYLWW 13 CEA 345 204 1027 3589 12 508 865 2 402 PEIQNTTYL 9 CEA 347 923 138 2786 16,816 231 1825 2 403 AELPKPSI 8 CEA 498 7423 6697 131 959 19 2608 2 404 PEAQNTTY 8 CEA 525 149 2594 2437 — 76 3255 2 405 AEGKEVLL 8 CEA 46 11,455 1311 5303 17,268 129 14,165 1 406 AEPPKPFIT 9 CEA 320 14,614 7067 3438 — 214 1838 1 407 CEPEIQNTT 9 CEA 345 8575 10,080 1453 19,027 119 2401 1 408 CEPEIQNTTY 10 CEA 345 1459 — — 49 14,596 — 1 409 PEIQNTTYLW 10 CEA 347 819 3301 9423 13 6173 10,011 1 410 QELFISNI 8 CEA 460 889 6396 1175 2282 70 1172 1 411 TEKNSGLY 8 CEA 468 211 9851 7117 1868 605 10,248 1 412 TEKNSGLYT 9 CEA 468 713 7522 1724 6134 99 1850 1 413 CEPEAQNTTY 10 CEA 523 9525 — — 61 — 17,330 1 414 CEPEAQNTTYL 11 CEA 523 962 2184 11,723 3419 131 2450 1 415 PEAQNTTYLW 10 CEA 525 17,082 — — 27 — — 1 416 NEEATGQF 8 CEA 131 7326 — — 8366 17,054 17,737 0 417 PELPKPSI 8 CEA 142 9106 — — — 5143 — 0 418 VEDKDAVAFT 10 CEA 157 1434 11,648 2077 16,919 803 — 0 419 CEPETQDATY 10 CEA 167 1353 — — 787 536 — 0 420 PETQDATY 8 CEA 169 10,803 — — 6343 19,466 — 0 421 PETQDATYL 9 CEA 169 13,219 1374 — — 2488 13,430 0 422 PETQDATYLW 10 CEA 169 9346 — — 850 915 — 0 423 PETQDATYLWW 11 CEA 169 5308 — — 2819 600 11,631 0 424 CETQNPV 7 CEA 215 5798 18,808 7450 13,463 819 — 0 425 AEPPKPFI 8 CEA 320 12,800 — — 13,720 904 — 0 426 VEDEDAVALT 10 CEA 335 7275 895 2359 6668 2682 — 0 427 PEIQNTTY 8 CEA 347 1023 7274 — — 1618 — 0 428 CEPEAQNTT 9 CEA 523 15,594 11,134 — — 1212 2579 0 429 PEAQNTTYL 9 CEA 525 9500 3092 9280 — 1840 — 0

TABLE 27 HER2/new-derived B44 peptides SEQ ID NO Sequence AA Protein Position B*1801 B*4001 B*4002 B*4402 B*4403 B*4501 Degeneracy 430 MELAALCRWGL 11 Her2/neu 1 6.4 24 30 17 0.92 116 6 431 LELTYLPTNASL 12 Her2/neu 60 88 38 31 420 3.7 37 6 432 QEVQGYVLI 9 Her2/neu 78 42 36 28 45 1.7 92 6 433 GEGLACHQLCA 11 Her2/neu 506 62 39 97 159 2.7 196 6 434 AEQRASPL 8 Her2/neu 644 16 73 13 243 0.38 120 6 435 AEQRASPLTSI 11 Her2/neu 644 467 19 58 5.1 2.5 11 6 436 KEILDEAYVM 10 Her2/neu 765 13 5.5 4.0 35 3.3 234 6 437 GERLPQPPI 9 Her2/neu 938 62 71 3.3 27 1.1 15 6 438 SECRPRFREL 10 Her2/neu 963 80 307 18 11 0.20 25 6 439 AENPEYLGL 9 Her2/neu 1243 17 2.2 271 45 2.5 155 6 440 RELQLRSLTEI 11 Her2/neu 138 261 2.8 3.7 125 0.99 269 6 441 QEFAGCKKIFG 11 Her2/neu 362 211 423 477 37 2.1 138 6 442 LEEITGYLYISA 12 Her2/neu 403 35 177 78 323 4.6 110 6 443 SEFSRMARDPQRF 13 Her2/neu 974 44 454 45 54 11 361 6 444 NEDLGPASPL 10 Her2/neu 991 107 281 150 40 6.0 231 6 445 SEEEAPRSPL 10 Her2/neu 1066 151 155 217 37 8.4 84 6 446 SETDGYVAPL 10 Her2/neu 1122 94 214 184 386 2.4 302 6 447 LELTYLPTNA 10 Her2/neu 60 332 325 10 6428 3.1 24 5 448 QEVQGYVL 8 Her2/neu 78 3.4 28 5.0 1307 0.92 33 5 449 FEDNYALAV 9 Her2/neu 108 9.5 11 6.2 9942 0.42 154 5 450 RELQLRSLT 9 Her2/neu 138 638 316 13 465 0.20 162 5 451 MEHLREVRA 9 Her2/neu 347 233 — 386 38 3.2 19 5 452 MEHLREVRAV 10 Her2/neu 347 72 — 160 180 13 140 5 453 MEHLREVRAVTSA 13 Her2/neu 347 77 5662 120 281 21 16 5 454 QEFAGCKKIF 10 Her2/neu 362 53 3686 12 4.0 3.6 115 5 455 EEITGYLYISA 11 Her2/neu 404 0.94 1440 52 4.5 2.1 0.93 5 456 RELGSGLAL 9 Her2/neu 459 359 3.7 0.9 473 0.97 2262 5 457 RELGSGLALI 10 Her2/neu 459 4810 22 4.4 32 0.78 324 5 458 GEGLACHQL 9 Her2/neu 506 13,766 14 88 66 11 340 5 459 REYVNARHCL 10 Her2/neu 552 1327 39 4.8 106 0.97 126 5 460 EEGACQPCPI 10 Her2/neu 619 119 — 340 52 80 401 5 461 AEQRASPLT 9 Her2/neu 644 346 874 183 103 1.8 10 5 462 QETELVEPL 9 Her2/neu 692 12 9.1 36 310 3.5 1232 5 463 QETELVEPLT 10 Her2/neu 692 15 293 338 1619 13 288 5 464 GENVKIPVAI 10 Her2/neu 743 563 314 3.7 230 2.8 198 5 465 RELVSEFSRM 10 Her2/neu 970 9.1 28 4.3 33 0.12 1726 5 466 RELVSEFSRMA 11 Her2/neu 970 168 191 143 2613 3.5 32 5 467 AEEYLVPQQGFF 12 Her2/neu 1020 124 262 589 24 49 172 5 468 SEDPTVPL 8 Her2/neu 1113 103 71 161 9450 2.0 308 5 469 SETDGYVAPLT 11 Her2/neu 1122 66 125 224 1225 2.2 45 5 470 AENPEYLGLDV 11 Her2/neu 1243 11,934 28 139 69 3.0 24 5 471 MELAALCRW 9 Her2/neu 1 7.5 4301 141 26 15 1140 4 472 MELAALCRWG 10 Her2/neu 1 102 8684 1840 5.7 135 408 4 473 QEVQGYVLIA 10 Her2/neu 78 61 772 64 1871 15 11 4 474 FEDNYALAVL 10 Her2/neu 108 321 6.2 48 2844 3.8 3095 4 475 RELQLRSL 8 Her2/neu 138 42 49 5.9 2248 0.62 1372 4 476 TEILKGGVL 9 Her2/neu 146 125 30 14 697 0.28 2480 4 477 TEILKGGVLI 10 Her2/neu 146 1021 241 294 24 21 7600 4 478 GESSEDCQSL 10 Her2/neu 206 — 8.1 23 427 5.1 2491 4 479 SEDCQSLTRTV 11 Her2/neu 209 101 4322 311 943 21 10 4 480 PEGRYTFGASCV 12 Her2/neu 285 1366 2.6 6.1 1410 348 356 4 481 REVRAVTSA 9 Her2/neu 351 626 427 0.7 3160 0.18 9.3 4 482 REVRAVTSANI 11 Her2/neu 351 4491 17 30 1680 1.8 421 4 483 EEITGYLY 8 Her2/neu 404 20 5713 1223 38 83 238 4 484 EEITGYLYI 9 Her2/neu 404 86 906 916 14 121 94 4 485 EEITGYLYISAW 12 Her2/neu 404 36 837 1966 37 87 57 4 486 QECVEECRVL 10 Her2/neu 538 315 444 399 606 22 2863 4 487 VEECRVLQGL 10 Her2/neu 541 270 227 5815 237 189 16,094 4 488 GENVKIPVA 9 Her2/neu 743 1508 293 3.0 — 1.7 13 4 489 KEILDEAYV 9 Her2/neu 765 1358 62 43 6466 8.4 42 4 490 KEILDEAYVMA 11 Her2/neu 765 731 252 95 11,514 64 123 4 491 DEAYVMAGVG 10 Her2/neu 769 122 203 154 4033 5609 218 4 492 TEYHADGGKVPI 12 Her2/neu 875 632 195 7.1 3342 1.4 361 4 493 GERLPQPPICTI 12 Her2/neu 938 8538 398 9.5 935 0.60 40 4 494 AEEYLVPQQGF 11 Her2/neu 1020 125 584 1831 21 99 268 4 495 EEYLVPQQG 9 Her2/neu 1021 66 10,344 176 2200 126 131 4 496 EEYLVPQQGF 10 Her2/neu 1021 12 — 2551 21 11 73 4 497 EEYLVPQQGFF 11 Her2/neu 1021 94 4291 1695 78 168 154 4 498 EEEAPRSPL 9 Her2/neu 1067 902 4490 316 177 362 307 4 499 EEAPRSPLA 9 Her2/neu 1068 486 10,707 4900 200 294 4.5 4 500 REGPLPAARPA 11 Her2/neu 1153 157 543 78 — 4.2 347 4 501 RELQLRSLTEIL 12 Her2/neu 138 1252 15 26 2286 0.50 865 3 502 GESSEDCQSLT 11 Her2/neu 206 742 48 180 14,386 40 2158 3 503 CELHCPAL 8 Her2/neu 264 150 871 259 4361 39 — 3 504 CELHCPALV 9 Her2/neu 264 136 4805 319 2308 52 1110 3 505 CELHCPALVT 10 Her2/neu 264 80 — 65 933 18 1275 3 506 CELHCPALVTY 11 Her2/neu 264 12 3469 3198 140 89 2779 3 507 FESMPNPEG 9 Her2/neu 279 6068 — 59 14,846 20 155 3 508 FESMPNPEGRY 11 Her2/neu 279 74 3666 3533 59 70 1394 3 509 QEFAGCKKI 9 Her2/neu 362 1120 736 131 85 44 2684 3 510 FETLEEITGYL 11 Her2/neu 400 133 78 649 7490 42 2200 3 511 LEEITGYLYI 10 Her2/neu 403 143 914 2996 222 143 1488 3 512 PEDECVGEGL 10 Her2/neu 500 1257 278 257 6331 49 — 3 513 DECVGEGL 8 Her2/neu 502 49 4864 481 938 34 14,244 3 514 TELVEPLTPSGA 12 Her2/neu 694 167 4104 103 2118 28 2739 3 515 RENTSPKANKEIL 13 Her2/neu 756 11,950 5.8 64 — 8.6 9105 3 516 KEILDEAY 8 Her2/neu 765 82 921 430 7485 74 2646 3 517 DEAYVMAGV 9 Her2/neu 769 58 5327 1245 8006 138 161 3 518 LESILRRRF 9 Her2/neu 891 29 — 3475 5.8 101 12,918 3 519 WELMTFGAKPY 11 Her2/neu 913 13 509 778 24 75 1216 3 520 REIPDLLEKGERL 13 Her2/neu 929 3212 34 226 2914 31 14,043 3 521 GERLPQPPICT 11 Her2/neu 938 12,486 — 23 9094 3.9 15 3 522 SECRPRFRELV 11 Her2/neu 963 1996 3673 121 927 18 118 3 523 SEGAGSDVF 9 Her2/neu 1078 74 5627 6525 33 192 6960 3 524 PEYLGLDVPV 10 Her2/neu 1246 613 352 35 1371 1.7 610 3 525 CEKCSKPCARVCY 13 Her2/neu 331 763 16,796 1292 340 117 1815 2 526 MEHLREVRAVT 11 Her2/neu 347 1064 1963 2207 795 111 74 2 527 LEEITGYL 8 Her2/neu 403 242 830 1805 8038 403 — 2 528 DEEGACQPCPI 11 Her2/neu 618 451 5517 7293 968 438 1323 2 529 TELVEPL 7 Her2/neu 694 162 14,164 1258 8854 66 — 2 530 VEPLTPSGA 9 Her2/neu 697 7321 — 96 8516 191 17,037 2 531 KETELRKVKV 10 Her2/neu 716 11,925 — 68 2936 15 1603 2 532 TELRKVKVL 9 Her2/neu 718 1514 4698 11 1844 2.5 14,147 2 533 LEDVRLVHRDL 11 Her2/neu 836 729 325 641 818 59 2382 2 534 TEYHADGGKV 10 Her2/neu 875 239 5246 2003 2911 15 1571 2 535 LESILRRRFT 10 Her2/neu 891 82 — 1189 34 657 2251 2 536 LEDDDMGDL 9 Her2/neu 1009 191 556 351 722 900 6251 2 537 AEEYLVPQQG 10 Her2/neu 1020 723 — — 1549 479 127 2 538 SEEEAPRSPLA 11 Her2/neu 1066 1318 3604 5110 8550 158 27 2 539 SEGAGSDVFDG 11 Her2/neu 1078 928 3751 5695 374 286 3008 2 540 PEYLTPQGGAA 11 Her2/neu 1194 1724 — 200 — 354 4011 2 541 PERGAPPST 9 Her2/neu 1228 390 4744 7679 1116 178 7767 2 542 PETHLDML 8 Her2/neu 39 1954 8387 6118 — 83 — 1 543 PETHLDMLRHL 11 Her2/neu 39 1322 700 2971 11,534 70 4329 1 544 SEDCQSL 7 Her2/neu 209 18,245 2691 14,258 8248 431 19,225 1 545 HEQCAAGCT 9 Her2/neu 237 1995 — 7377 14,068 178 2974 1 546 PEGRYTFGASCVT 13 Her2/neu 285 6602 4411 3286 1560 456 1198 1 547 QEVTAEDGT 9 Her2/neu 320 5207 — 3122 7886 66 1843 1 548 CEKCSKPCA 9 Her2/neu 331 3740 — 2703 12,538 342 8007 1 549 CEKCSKPCARV 11 Her2/neu 331 1167 4103 2079 9594 101 1561 1 550 REVRAVT 7 Her2/neu 351 8564 3136 725 — 29 — 1 551 FETLEEI 7 Her2/neu 400 1518 7621 2110 — 69 — 1 552 FETLEEITGY 10 Her2/neu 400 671 — — 262 1679 — 1 553 DECVGEGLACHQL 13 Her2/neu 502 586 4421 3965 3093 468 5888 1 554 QECVEECRV 9 Her2/neu 538 15,799 8755 1664 4348 210 4542 1 555 VEECRVLQG 9 Her2/neu 541 1528 8947 7622 12,736 305 — 1 556 EECRVLQGL 9 Her2/neu 542 890 7076 2029 717 434 1185 1 557 PECQPQNGSV 10 Her2/neu 565 7962 — — 12,964 472 — 1 558 TELVEPLTPSG 11 Her2/neu 11 694 601 2978 3703 — 269 14,079 1 559 VEPLTPSGAM 10 Her2/neu 697 4649 1667 584 4368 108 — 1 560 KETELRKVKVL 11 Her2/neu 716 9529 2973 1868 7136 71 12,237 1 561 TELRKVKVLG 10 Her2/neu 718 721 — 601 3650 14 12,816 1 562 DETEYHADG 9 Her2/neu 873 159 — — — 1397 13,353 1 563 DETEYHADGG 10 Her2/neu 873 613 — 16,801 3891 269 — 1 564 REIPDLLEKG 10 Her2/neu 929 649 4493 814 1270 13 1977 1 565 SECRPRF 7 Her2/neu 963 926 18,181 1157 852 48 8856 1 566 EEEAPRSPLA 10 Her2/neu 1067 6611 — — 3128 960 14 1 567 EEAPRSPL 8 Her2/neu 1068 1191 3489 1611 3020 171 1926 1 568 SEGAGSDVFDGDL 13 Her2/neu 1078 1563 52 2097 1595 749 3001 1 569 PEYVNQPDV 9 Her2/neu 1137 831 3437 1581 823 48 2536 1 570 VENPEYLTPQG 11 Her2/neu 1191 8386 — — 17,337 11 4188 1 571 PEYLTPQGG 9 Her2/neu 1194 1456 18,951 13,860 6532 284 18,990 1 572 PERGAPPSTF 10 Her2/neu 1228 1062 14,884 3437 6871 208 15,700 1 573 PETHLDM 7 Her2/neu 39 — 15,506 — — 5081 — 0 574 SEDCQSLTRT 10 Her2/neu 209 7257 8550 11,529 518 2857 5178 0 575 HEQCAAGCTG 10 Her2/neu 237 3805 6126 8285 3168 806 2072 0 576 PEGRYTFGA 9 Her2/neu 285 5252 — — — 658 15,119 0 577 PESFDGDPA 9 Her2/neu 378 14,489 11,550 — — 1170 4418 0 578 PEQLQVFET 9 Her2/neu 394 1798 11,635 — 5814 4659 9322 0 579 PEQLQVFETL 10 Her2/neu 394 1314 — 4890 6107 754 — 0 580 PEQLQVFETLEEI 13 Her2/neu 394 9147 5393 — — 3232 — 0 581 FETLEEITG 9 Her2/neu 400 1750 14,182 8715 10,330 753 — 0 582 LEEITGY 7 Her2/neu 403 8594 17,584 11,496 — 989 — 0 583 LEEITGYLY 9 Her2/neu 403 1048 13,469 — 870 5335 — 0 584 RILHNGAYSL 10 Her2/neu 434 2345 — 726 — — — 0 585 PEDECVGEG 9 Her2/neu 500 11,794 931 647 7327 559 — 0 586 PEDECVGEGLA 11 Her2/neu 500 6685 — — 4217 1933 — 0 587 DECVGEGLA 9 Her2/neu 502 1006 4742 3131 14,445 506 1114 0 588 PECQPQNGSVT 11 Her2/neu 565 8882 — — 5093 2353 — 0 589 PECQPQNGSVTCF 13 Her2/neu 565 4787 1772 8115 3564 2161 — 0 590 PEADQCVACA 10 Her2/neu 579 10,271 — 16,262 6242 2155 1486 0 591 PEADQCVACAHY 12 Her2/neu 579 2819 12,352 11,420 1163 3516 3057 0 592 LEDVRLV 7 Her2/neu 836 16,473 17,438 — — 2658 — 0 593 DETEYHA 7 Her2/neu 873 6105 — 18,324 — 10,523 — 0 594 LEDDDMGDLV 10 Her2/neu 1009 9176 2963 3596 6746 3641 10,475 0 595 LERPKTLSPG 10 Her2/neu 1167 10,194 — — 2333 1400 7162 0 596 VENPEYL 7 Her2/neu 1191 — 14,701 — 14,253 12,085 — 0 597 PEYLTPQGGA 10 Her2/neu 1194 7436 — — 9016 5779 15,093 0

TABLE 28 p53-derived B44 peptides SEQ ID NO Sequence AA Protein Position B*1801 B*4001 B*4002 B*4402 B*4403 B*4501 Degeneracy 598 RERFEMFREL 10 p53 335 83 29 17 17 0.34 422 6 599 GEYFTLQIRG 10 p53 325 308 88 19 2452 3.9 157 5 600 QETFSDLWKL 10 p53 16 736 199 255 39 14 901 4 601 DEAPRMPEA 9 p53 61 84 297 4577 6448 98 10 4 602 HERCSDSDGL 10 p53 179 139 171 61 1468 6.0 1723 4 603 VEYLDDRNTF 10 p53 203 0.94 501 37 32 1.4 3601 4 604 FEVRVCACPG 10 p53 270 64 2043 4.9 180 0.76 1872 4 605 GEYFTLQI 8 p53 325 7774 112 60 3511 1.0 261 4 606 FEMFRELNEA 10 p53 338 127 3207 223 952 2.0 208 4 607 FEMFRELNEAL 11 p53 338 475 17 8.8 748 1.1 1352 4 608 RELNEALEL 9 p53 342 3000 35 30 256 1.1 3337 4 609 IEQWFTEDPG 10 p53 50 151 1250 2114 5595 142 197 3 610 VEGNLRVEYL 10 p53 197 104 481 2565 1963 22 15,189 3 611 GEPHHELPPG 10 p53 293 108 3323 1888 11,728 4.4 20 3 612 TEDPGPDEAPRM 12 p53 55 570 361 1326 2791 141 1702 2 613 DEAPRMPEAA 10 p53 61 121 1497 8444 2594 1037 100 2 614 HERCSDSDG 9 p53 179 1118 67 — 2032 208 13,390 2 615 HERCSDSDGLA 11 p53 179 1408 4879 1915 — 96 186 2 616 LEDSSGNL 8 p53 257 17,736 782 108 — 211 15,946 2 617 LEDSSGNLL 9 p53 257 1140 2.2 2771 1865 43 — 2 618 GEPHHELPPGST 12 p53 293 3814 — 5418 4477 413 132 2 619 RERFEMF 7 p53 335 180 4079 1907 — 108 — 2 620 LELKDAQAG 9 p53 348 170 18,706 3659 5126 30 1989 2 621 MEEPQSDPSV 10 p53 1 8970 3802 16,536 1927 816 175 1 622 VEPPLSQET 9 p53 10 8302 17,052 — 3186 236 — 1 623 VEPPLSQETF 10 p53 10 814 — — 406 525 — 1 624 QETFSDLWKLL 11 p53 16 4158 3366 740 631 168 1218 1 625 PENNVLSPL 9 p53 27 1150 1261 718 11,174 8.8 — 1 626 DEAPRMPEAAPPV 13 p53 61 583 — 2715 — 1727 87 1 627 VEGNLRVEY 9 p53 197 832 12,752 — 61 2583 — 1 628 VEYLDDRNT 9 p53 203 1442 — — 10,071 157 13,503 1 629 YEPPEVGSDCT 11 p53 220 16,872 — 125 13,349 12,712 19,199 1 630 YEPPEVGSDCTTI 13 p53 220 9330 3530 689 3009 351 — 1 631 PEVGSDCTTI 10 p53 223 611 4552 248 2293 2046 — 1 632 LEDSSGNLLG 10 p53 257 1062 531 697 7905 153 19,256 1 633 TEEENLRKKG 10 p53 284 — — — — 315 — 1 634 HELPPGSTKRA 11 p53 297 6034 3974 3255 — 189 1472 1 635 NEALELKDA 9 p53 345 1925 3887 6640 4270 1582 129 1 636 NEALELKDAQA 11 p53 345 742 6235 5071 — 949 53 1 637 EEPQSDPSV 9 p53 2 9454 — — 5972 1507 1620 0 638 QETFSDL 7 p53 16 8514 18,350 12,210 — 1612 18,051 0 639 TEDPGPDEA 9 p53 55 15,049 1217 — 8035 2763 1789 0 640 PEAAPPV 7 p53 67 14,223 — — — 5429 — 0 641 PEAAPPVAPA 10 p53 67 2638 5800 14,593 2192 653 901 0 642 PEVGSDCTT 9 p53 223 18,612 1189 — 7109 12,997 — 0 643 PEVGSDCTTIHY 12 p53 223 1182 10,188 7994 4093 1939 2251 0 644 EEENLRKKG 9 p53 285 1464 687 — 3662 706 18,421 0

TABLE 29 MAGE2-derived B44 peptides SEQ ID NO Sequence AA Protein Position B*1801 B*4001 B*4002 B*4402 B*4403 B*4501 Degeneracy 645 LESEFQAAI 9 MAGE2 101 14 41 39 43 1.0 78 6 646 LESEFQAAISRKM 13 MAGE2 101 26 264 46 427 14 102 6 647 SEFQAAISRKM 11 MAGE2 103 7.0 345 107 88 1.2 161 6 648 SEFQAAISRKMV 12 MAGE2 103 47 300 25 111 3.2 256 6 649 SEYLQLVFG 9 MAGE2 155 18 235 421 348 19 113 6 650 SEYLQLVFGI 10 MAGE2 155 5.2 20 6.1 3.7 0.84 4.4 6 651 SEYLQLVFGIEVV 13 MAGE2 155 12 44 17 229 7.6 22 6 652 WEELSMLEVF 10 MAGE2 222 4.0 463 30 15 22 290 6 653 QENYLEYRQV 10 MAGE2 252 210 493 102 17 16 27 6 654 YEFLWGPRALI 11 MAGE2 269 5.2 4.1 2.8 92 0.59 450 6 655 LEARGEALGLVGA 13 MAGE2 16 228 29 50 3886 3.7 135 5 656 VEVTLGEVPA 10 MAGE2 46 14 371 31 3801 0.52 15 5 657 EEGPRMFPDL 10 MAGE2 92 128 4438 486 291 13 42 5 658 AEMLESVL 8 MAGE2 133 968 14 31 327 0.88 302 5 659 LESVLRNCQDFF 12 MAGE2 136 56 246 370 264 54 1466 5 660 SEYLQLVF 8 MAGE2 155 0.97 765 6.0 284 0.70 122 5 661 VEVVPISHLYI 11 MAGE2 167 97 135 146 335 7.2 3788 5 662 EEKIWEELSM 10 MAGE2 218 86 — 477 46 28 107 5 663 EELSMLEVFEG 11 MAGE2 223 1.5 — 294 4.6 23 163 5 664 YEFLWGPRA 9 MAGE2 269 5.3 30 5.2 4246 1.1 241 5 665 YEFLWGPRAL 10 MAGE2 269 17 8.5 1.6 130 0.72 753 5 667 IETSYVKVL 9 MAGE2 279 72 7.2 23 33 2.6 11,902 5 668 LEARGEAL 8 MAGE2 16 163 99 26 — 2.9 — 4 669 LEARGEALGL 10 MAGE2 16 81 184 277 2275 4.1 3046 4 670 LEARGEALGLV 11 MAGE2 16 158 198 345 — 13 1912 4 671 VELVHFLL 8 MAGE2 114 5.0 69 31 3322 1.2 2427 4 672 VELVHFLLL 9 MAGE2 114 71 79 31 559 3.1 1129 4 673 REPVTKAEM 9 MAGE2 127 60 40 284 6577 4.5 832 4 674 REPVTKAEML 10 MAGE2 127 88 23 264 763 21 917 4 675 IEVVEVVPI 9 MAGE2 164 11 4.7 60 11,313 1.3 6423 4 676 VEVVPISHL 9 MAGE2 167 149 2.8 66 9082 2.3 13,803 4 678 VEVVPISHLYIL 12 MAGE2 167 191 20 17 935 3.2 1926 4 679 VEVVPISHLYILV 13 MAGE2 167 197 373 110 562 25 839 4 680 WEELSMLEV 9 MAGE2 222 70 2174 37 657 2.5 134 4 681 EELSMLEVF 9 MAGE2 223 1.4 16,436 252 22 2.8 1013 4 682 GEPHISYPPL 10 MAGE2 295 7254 7.0 2.9 1200 0.71 380 4 683 EEGLEARGEAL 11 MAGE2 13 179 300 578 2630 19 1812 3 684 GEALGLVGA 9 MAGE2 20 9529 510 34 6134 2.2 17 3 685 GEALGLVGQA 11 MAGE2 20 877 4293 52 3575 1.4 28 3 686 EEQQTASSSSTL 12 MAGE2 34 8999 301 2287 160 570 205 3 687 QEEEGPRM 8 MAGE2 90 298 11,598 431 19,255 118 6730 3 688 QEEEGPRMF 9 MAGE2 90 414 626 7747 237 409 2171 3 689 VELVHFLLLKY 11 MAGE2 114 52 550 294 1551 49 1790 3 690 LESVLRNCQDF 11 MAGE2 136 64 5409 3458 209 76 15,241 3 691 IEVVEVVPISHLY 13 MAGE2 164 108 1191 610 214 123 2639 3 692 VEVVPISHLY 10 MAGE2 167 99 11,522 4385 13 346 6776 3 693 FEGREDSVF 9 MAGE2 231 9.8 2366 348 4434 13 3339 3 694 EEGLEARGEA 10 MAGE2 13 1077 3434 3227 216 684 30 2 695 LEARGEALG 9 MAGE2 16 155 1161 3006 — 24 2688 2 696 VEVTLGEVPAA 11 MAGE2 46 124 — 919 — 44 1583 2 697 EEEGPRMFPDL 11 MAGE2 91 1011 2646 3470 3273 131 209 2 698 SEFQAAI 7 MAGE2 103 181 6830 779 2660 33 9597 2 699 REPVTKAEMLESV 13 MAGE2 127 2495 253 605 4546 40 4579 2 700 IEGDCAPEEKI 11 MAGE2 211 844 — — 2627 486 183 2 701 EEKIWEEL 8 MAGE2 218 753 9084 2599 12,420 104 171 2 702 EEKIWEELSML 11 MAGE2 218 1641 4978 — 1862 375 181 2 703 LEVFEGREDSV 11 MAGE2 228 639 2624 295 — 46 — 2 704 FEGREDSVFA 10 MAGE2 231 242 — 4775 6879 192 503 2 705 PEEGLEARG 9 MAGE2 12 1252 292 — — 2094 1967 1 706 EEQQTASSSST 11 MAGE2 34 752 2306 — 5910 1552 134 1 707 QEEEGPRMFPDL 12 MAGE2 90 4178 1769 2931 2186 394 1200 1 708 EEEGPRMF 8 MAGE2 91 723 12,281 — 2406 213 943 1 709 SEYLQLV 7 MAGE2 155 1375 7777 658 733 21 930 1 710 PEEKIWEEL 9 MAGE2 217 577 19,449 3908 2893 235 17,345 1 711 GEPHISY 7 MAGE2 295 8833 12,272 6716 — 272 — 1 712 PEEGLEARGEA 11 MAGE2 12 15,209 — 18,624 — 950 3193 0 713 EEGLEARGEALGL 13 MAGE2 13 8199 3694 18,543 2515 4468 5856 0 714 DEGSSNQEEEG 11 MAGE2 84 10,836 — — — 2125 — 0 715 PEEKIWEELSM 11 MAGE2 217 6039 — 18,680 — 985 15,500 0 716 WEELSML 7 MAGE2 222 1288 781 740 — 610 — 0 717 WEELSML 7 MAGE2 234 17,670 10,408 — — 2010 15,074 0 718 WEELSML 7 MAGE2 252 8292 6581 — 2999 10,009 2724 0

TABLE 30 MAGE3-defived B44 SEQ ID NO Sequence AA Protein Position B*1801 B*4001 B*4002 B*4402 B*4403 B*4501 Degeneracy 719 QEAASSSSTL 10 MAGE2 36 144 49 47 56 13 287 6 720 LESEFQAAL 9 MAGE3 301 5.4 19 16 95 1.0 113 6 721 AELVHFLLL 9 MAGE3 114 160 25 3.1 18 0.94 141 6 722 MEVDPIGHL 9 MAGE3 167 14 1.6 21 165 1.7 247 6 723 MEVDPIGHLYI 11 MAGE3 167 9.8 34 16 64 0.91 95 6 724 AELVHFLL 8 MAGE3 114 120 71 6.8 1186 0.16 452 5 725 AELVHFLLLKY 11 MAGE3 114 153 32 39 178 1.6 670 5 726 AEMLGSVVG 9 MAGE3 133 96 1899 109 19 1.6 11 5 727 EEKIWEELSV 10 MAGE3 218 449 8947 79 396 17 17 5 728 WEELSVLEVF 10 MAGE3 222 14 75 37 14 13 1701 5 729 VETSYVKVL 9 MAGE3 279 87 7.8 57 80 1.1 2687 5 730 EEGPSTFPDL 10 MAGE3 92 165 655 591 198 127 128 4 731 IELMEVDPI 9 MAGE3 164 78 296 252 4042 3.1 11,937 4 732 MEVDPIGHLY 10 MAGE3 167 14 617 625 11 99 169 4 733 EEKIWEELSVL 11 MAGE3 218 133 25 1255 1416 58 218 4 734 EELSVLEVF 9 MAGE3 223 7.3 75 3314 99 12 2120 4 735 EEEGPSTF 8 MAGE3 91 201 1008 435 3933 27 1819 3 736 EEEGPSTFPDL 11 MAGE3 91 935 431 2120 2685 102 158 3 737 WEELSVLEV 9 MAGE3 222 8.0 2479 158 — 2.6 538 3 738 FEGREDSIL 9 MAGE3 231 1091 4.9 439 1925 11 — 3 739 FEGREDSILG 10 MAGE3 231 229 460 4361 8534 172 — 3 740 QEAASSSST 9 MAGE3 36 1422 — 1480 3823 41 110 2 741 REGDCAPEEKI 11 MAGE3 211 973 2418 830 4038 42 146 2 742 LEVFEGREDSI 11 MAGE3 228 4745 206 512 — 69 — 2 743 FEGREDSI 8 MAGE3 231 5763 718 127 14,181 13 2291 2 744 QEEEGPSTF 9 MAGE3 90 841 — 16,118 324 529 2450 1 745 IELMEVDPIG 10 MAGE3 164 506 6592 5325 222 — 7604 1

TABLE 31 Hepatitis B Virus Core Protein (SEQ ID NO:754) MQLFHLLIISCSCPTVQASKLCLGWLWGMDIDPYKEFGATVELLSFLPSDFFPSV RDLLDTASALYREALESPEHCSPHHTALRQAILCWGELMTLATWVGVNLEDPASR DLVVSYVNTNMGLKFRQLLWFHISCLTFGRETVIEYLVSFGVWIRTPPAYRPPNAPIL STLPETTVVRRRGRSPRRRTPSPRRRRSQSPRRRRSQSRESQC

TABLE 32 Population coverage by allelic family Phenotypic frequencies by supertype family Minimal Population Coverage Allelle Cauc. Blk. Jpn. Chn. His. Avg. A2 sup 45.8 39.0 42.4 45.9 43.0 43.2 A3 sup 37.5 42.1 45.8 52.7 43.1 44.2 B7 sup 38.6 52.7 48.8 35.5 47.1 44.7 A1 specific 28.6 10.1  1.4  9.2 10.1 11.9 A24 specific 16.8  8.8 58.1 32.9 26.7 28.7 Scenarios: A A3 super, B7 super, A1 & A24 B A3 super, B7 super, A1, A24 & A2 super C A3 super, A1 & A24 D A3 super, A1, A24 & A2 super Phenotypic frequencies of combined supertype families Minimal Population Coverage Scenario Cauc. Blk. Jpn. Chn. His. Avg. A 81.6 79.1 92.7 86.4 83.5 84.7 B 95.1 90.6 99.1 97.5 94.8 95.4 C 70.0 55.9 85.8 78.9 68.8 71.9 D 92.0 80.2 98.2 96.2 90.1 91.3 Phenotypic frequencies by supertype family Minimal Population Coverage Allelle Cauc. Blk. Jpn. Chn. His. Avg. A3 super 37.5 42.1 45.8 52.7 43.1 44.2 B7 super 38.6 52.7 48.8 35.5 47.1 44.7 A1 specific 28.6 10.1 1.4 9.2 10.1 11.9 A24 specific 16.8 8.8 58.1 32.9 26.7 28.7 Scenarios: A A3 super & B7 super B A1 & A24 C A3 super, A1 & A24 D B7 super, A1 & A24 E A3 super, B7 super, A1 & A24 Phenotypic frequencies of combined supertype families Minimal Population Coverage Scenario Cauc. Blk. Jpn. Chn. His. Avg. A 61.6 72.6 72.3 69.5 69.9 69.2 B 42.7 18.4 59.0 40.4 35.3 39.2 C 70.0 55.9 85.8 78.9 68.8 71.9 D 64.8 61.4 79.0 61.5 65.8 66.5 E 81.6 79.1 92.7 86.4 83.5 84.7 Phenotypic frequencies by supertype family Minimal Population Coverage Allelle Cauc. Blk. Jpn. Chn. His. Avg. A2 sup 45.8 39.0 42.4 45.9 43.0 43.2 A3 sup 37.5 42.1 45.8 52.7 43.1 44.2 B7 sup 38.6 52.7 48.8 35.5 47.1 44.7 A1 specific 28.6 10.1 1.4 9.2 10.1 11.9 A24 specific 16.8 8.8 58.1 32.9 26.7 28.7 A1 super 47.1 16.1 21.8 14.7 26.3 25.2 A24 super 23.9 38.9 58.6 40.1 38.3 40.0 Scenarios: A A2 super, A3 super, B7 super B A2 super, A3 super, A1, A24 C A2 super, A3 super, A1, A24, B7 super D A2 super, A3 super, A1 super, A24 super E A2 super, A3 super, A1 super, A24 super, B7 super Phenotypic frequencies of combined supertype families Minimal Population Coverage Scenario Cauc. Blk. Jpn. Chn. His. Avg. A 83.0 86.1 87.5 88.4 86.3 86.2 B 92.0 80.2 98.2 96.2 90.1 91.3 C 95.1 90.6 99.1 97.5 94.8 95.4 D 98.4 94.2 99.9 98.5 97.6 97.8 E 99.0 97.3 100.0 99.1 98.8 98.8

TABLE 33 Examples of Fragments CEA Fragments SEQ ID NO:754 (fragment comprising at least SEQ ID NO:161 (bold/underlined)) MESPSAPPHRWCIPWQRLLLTASLLTFWNPPITAKLTIESTPFNVAEGKEVLLLVHNL PQHLFGYSWYKGERVDGNRQIIGYVIGTQQATPGPAYSGREIIYPNASLLIQNIIQNDT GFYTLHVIKSDLVNEEATGQFRVYPELPKPSISSNNSKPVEDKDAVAFTCEPETQDAT YLWWVNNQSLPVSPRLQLSNGNRTLTLFNVTRNDTASYKCETQNPVSAR RSDSVILN VLY GPDAPTISPLNTSYRSGENLNLSCHAASNPPAQYSWFVNGTFQQSTQELFIPNITV NNSGSYTCQAHNSDTGLNRTTVTTITVYAEPPKPFITSNNSNPVEDEDAVALTCEPEIQ NTTYLWWVNNQSLPVSPRLQLSNDNRTLTLLSVTRNDVGPYECG SEQ ID NO:755 (fragment comprising at least SEQ ID NO:182 (bold/underlined)) PDSSYLSGANLNLSC HSANSNPSPQY SWRINGIPQQHTQVLFIAKITPNNNGTYACFVSN LATGRNNSIVKSITVSASGTSPGLSAGATVGIMIGVLVGVALI SEQ ID NO:756 (fragment comprising at least SEQ ID NO:181 (bold/underlined)) SAN RSDPVTLDVLY GPDTP HER2/neu Fragments SEQ ID NO:757 (fragment comprising at least SEQ ID NO:189 (bold/underlined)) MPNQAQMRILKETELRKVKVLGSGAFGTVYKGIWIPDGENVKIPVAIKVLRENTSPKA NKEILDEAYVMAGVGSPYVSRLLGICLTSTVQLVTQLMPYGCLLDHVRENRGRLGSQ DLLNWCMQIAKGMSYLEDVRLVHRDLAARNVLVKSPNHVKITDFGLAR LLDIDETE Y HADGGKVPIKWMALESILRRRFTHQSDVWSYGVTVWELMTFGAKPYDGIPAREIPD LLEKGERLPQPPICTIDVYMIMVKCWMIDSECRPRFRELVSEFSRMARDPQRFVVIQN EDLGPASPLDSTFYRSLLEDDDMGDLVDAEEYLVPQQGFFCPDPAPGAGGMVHHRH RSSSTRSGGGDLTLGLEPSEEEAPRSPLAPSEGAGSDVFDGDLGMGAAKGLQSLPTHD PSPLQRYSEDPTVPLPSETDGYVAPLTCSPQPEYVNQPDVRPQPPSPREGPLPAARPAG ATLERPKTLSPGKNGVVKDVFAFGGAVENPEYLTPQGGAAPQPHPPPAFSPAFDNLY YWDQDPPERGAPPSTFKGTPTAENPEYLGLDVPV SEQ ID NO:758 (fragment comprising at least SEQ ID NO:201 (bold/underlined)) MVHHRHRSSSTRSGGGDLTLGLEPSEEEAPRSPLAPSEGAGSDVFDGDLGMGAAKGL PAARPAGATLERPKTLSPGKNGVVKDVFAFGGAVENPEYLTPQGGAAPQPHPPPA FS PAFDNLYY WDQDPPERGAPPSTFKGTPTAENPEYLGLDVPV SEQ ID NO:759 (fragment comprising at least SEQ ID NO:191 (bold/underlined)) MALESILRRR FTHQSDVWSY GVTVWEL MAGE-2/3 Fragments SEQ ID NO:760 (MAGE-2 fragment comprising at least SEQ ID NO:219 (bold/underlined)) MPKTGLLIIVLAIIAIEGDCAPEEKIWEELSMLEVFEGREDSVFAHPRKLL MQDLVQEN Y LEYRQVPGSDPACYEFLWGPRALIETSYVKVLHHTLKIGGEPHISTPPLHERALREGE E SEQ ID NO:761 (MAGE-3 fragment comprising at least SEQ ID NO:221 (bold/underlined)) MPLEQRSQHCKPEEGLEARGEALGLVGAQAPATEEQEAASSSSTLVEVTLGEVPAAE SPDPPQSPQG ASSLPTTMNY PLWSQSYEDSSNQEEEGPSTFPDLESEFQAALSRKVAE LVHFLLLKYRAREPVTKAEMLGSVVGNWQYFFPVIFSKASSSLQLVFGIEL SEQ ID NO:762 (MAGE-3 fragment comprising at least SEQ ID NO:233 (bold/underlined)) KL LTQHFVQENY LEY p53 Fragments SEQ ID NO:763 (fragment comprising at least SEQ ID NO:239 (bold/underlined)) MDDLMLSPDDIEQWFTEDPGPDEAPRMPEAAPPVAPAPAAPTPAAPAPAPSWPLSSSV PSQKTYQGSYGFRLGFLHS GTAKSVTCTY SPALNKMFCQLAKTCPVQLWVDSTPPP GTRVRAMAIYKQSQHMTEVVRRCPHHERCSDSDGLAPPQHLIRVEGNLRVEYLDDR NTFRHSVVVPYEPPEVGSDCTTIHYNYMCNSSCMGGMNRRPILTIITLEDSSGNLLGR NSFEVRVCACPGRDRRTEEENLRKKGEPHHELPPGSTKRALPNNTSSSPQPKKKPLDG EYFTLQIRGRERFEMFRELNEALELKDAQAGKEPGGSRAHSSHLKSKKGQSTSRHKK LMFKTEGPDSD SEQ ID NO:764 (fragment comprising at least SEQ ID NO:239 (bold/underlined)) HS GTAKSVTCTY SPALNKM SEQ ID NO:765 (fragment comprising at least SEQ ID NO:244 (bold/underlined)) MFCQLAKTCPVQLWVDSTPPPGTRVRAMAIYKQSQHMTEVVRRCPHHERCSDSDGL APPQHLIRVEGNLRVEYLDDRNTFRHSVVVPYEPPEV GSDCTTIHY 

1-31. (canceled)
 32. An isolated peptide less than about 50 amino acids in length, wherein said peptide comprises an epitope or analog derived from a tumor associated antigen, and wherein said antigen is selected from the group consisting of: carcinoembryonic antigen (CEA), p53, HER2/neu, MAGE 2 and MAGE
 3. 33. A composition comprising the peptide of claim 32 and a carrier.
 34. The composition of claim 33, further comprising a CTL-inducing peptide.
 35. The composition of claim 34, wherein said CTL-inducing peptide also comprises an epitope or analog derived from a tumor associated antigen selected from the group set out in claim
 32. 36. The composition of claim 33, further comprising a T helper peptide.
 37. The composition of claim 36, wherein said T helper peptide is a pan-DR binding epitope.
 38. A composition comprising at least three peptides, wherein each of said peptides comprises an epitope or analog derived from a tumor associated antigen selected from the group set out in claim
 32. 39. The composition of claim 33, further comprising a liposome, wherein said peptide is on or within said liposome.
 40. The composition of claim 33, further comprising a lipid.
 41. The composition of claim 33, wherein said peptide is linked to a spacer molecule.
 42. The composition of claim 33, further comprising an antigen presenting cell, wherein said peptide is on or within said antigen presenting cell.
 43. The composition of claim 42, wherein said antigen presenting cell is a dendritic cell.
 44. A polypeptide comprising at least a first epitope and a second epitope, wherein said first epitope comprises an epitope or analog derived from a tumor associated antigen selected from the group set out in claim
 32. 45. A composition comprising the polypeptide of claim 44, wherein each of said first epitope and said second epitope comprises an epitope or analog derived from a tumor associated antigen selected from the group set out in claim
 32. 46. A composition comprising the polypeptide of claim 44, wherein said polypeptide is a homopolymer.
 47. A composition comprising the polypeptide of claim 44, wherein said polypeptide is a heteropolymer.
 48. An isolated nucleic acid encoding the peptide of claim
 32. 49. An isolated nucleic acid encoding the polypeptide of claim
 44. 50. A method of treating or preventing cancer in a mammal comprising administering the peptide of claim 32 to a mammal in need thereof.
 51. A method of treating or preventing cancer in a mammal comprising administering the nucleic acid of claim 48 to a mammal in need thereof. 